1. Using beta-escin and ionomycin-treated skinned smooth muscle strips of the rabbit mesenteric artery, the effects of calyculin A (CL-A, an inhibitor of type 1 and 2A phosphatases) on mechanical activities, phosphorylation of myosin light chain (MLC) and the relationship between the two were studied in Ca(2+)-free solution containing 4 mM EGTA and these effects were compared with those evoked by Ca2+. 2. The threshold concentration of Ca2+ required to increase either tension or MLC-phosphorylation was 0.1 microM and maximum effects were obtained at 10 microM. MLC was mainly monophosphorylated, rather than diphosphorylated, in the presence of Ca2+. ED50 value for Ca2+ was 0.54 microM for either tension or MLC-phosphorylation. The relationship between tension and MLC-phosphorylation is linear in the pCa range 7-5.5. 3. In Ca(2+)-free solution (containing either 20 mM EGTA or 4 mM EGTA with or without 4 mM BAPTA), 3 microM CL-A produced a contraction, the maximum amplitude of which was similar to that evoked by 10 microM Ca2+. CL-A (0.03-3 microM) concentration-dependently increased both tension and MLC-phosphorylation in Ca(2+)-free solution containing 4 mM EGTA. The threshold concentration of CL-A required for the increase in either tension or MLC-phosphorylation was 0.03 microM and maximum effects were obtained at 3 microM. In the presence of CL-A, MLC was not only monophosphorylated but also diphosphorylated. ED50 values for CL-A were 0.39 microM for tension, 0.44 microM for the monophosphorylated form of MLC and 0.54 microM for all phosphorylated (mono + di) forms. The relationship between tension and the monophosphorylated form of MLC was linear over the concentration range studied and was similar to that for Ca2+. 4. H-7 (3 microM, an inhibitor of protein kinase C) inhibited neither the tension nor phosphorylation of MLC induced by 10 microM Ca2+ or 3 microM CL-A. At a high concentration (30 microM), H-7 slightly inhibited both the tension and phosphorylation of MLC induced by either stimulant without a change in the tension-MLC-phosphorylation relationship. KN-62, an inhibitor of Ca(2+)-calmodulin-dependent protein kinase II, did not modify either the tension or the phosphorylation of MLC induced by 10 microM Ca2+ or 3 microM CL-A. CK-II, another inhibitor of Ca(2+)-calmodulin-dependent protein kinase II, did not inhibit the contraction induced by 3 microM CL-A. 5. SM-1 (0.03-0.3 mM) and ML-9 (0.1 and 0.3 mM), inhibitors of MLC-kinase, each lowered the resting level of MLC-phosphorylation in Ca2+-free solution and also inhibited both the tension and MLC-phosphorylation induced by 10 microM Ca2+ or 3 microM CL-A, in a concentration-dependent manner.Neither SM-1 nor ML-9 modified the relationship between tension and either monophosphorylated or all phosphorylated (mono + di) forms of MLC in the presence of Ca2+ or CL-A.6. In a solution containing MgITP (the substrate for myosin ATPase but not for MLC-kinase) with no MgATP, 10 microM Ca2+ failed to produce contraction. Under these conditions, the amplitude of the contraction induced by 3 microM CL-A was greatly diminished in comparison with that induced in the presence of MgATP.7. The present results suggest that in smooth muscle cells of the rabbit mesenteric artery, CL-A in Ca2+-free solution, produces a maximum contraction through an indirect activation of Ca2+-calmodulin independent(constitutively active) MLC-kinase via its inhibitory action on MLC-phosphatases. Based on this evidence, it is hypothesized that, in these cells, a constitutively active MLC-kinase may be present, though its action may be concealed by that of endogenous MLC-phosphatase.