We provide evidence for an immediate effect of NG-monomethyl-L-arginine (L-NMMA) on the force-frequency relationship in isolated hamster papillary muscles. L-NMMA (competitive inhibitor of nitric oxide synthase) reversed the force-frequency relationship (staircase effect) in isolated hamster papillary muscles from negative to positive (P < .01; ANOVA; n = 6). The addition of L-arginine (substrate for nitric oxide synthase) blocked the L-NMMA effect (P < .01; ANOVA; n = 6). The addition of the nitric oxide (NO) donor, sodium nitroprusside (NTP), significantly increased the level of cGMP in the tissue bath (P < .01; test; n = 6) and reversed the positive inotropic effect of L-NMMA on staircase (P < .01; ANOVA; n = 6). The addition of 8-Br-cGMP to the bath resulted in a concentration-dependent decrease in tension generated by the papillary muscles (n = 6). Methylene blue (known inhibitor of cGMP) mimicked the effect of L-NMMA on staircase (P < .01; ANOVA; n = 6). L-NMMA also significantly blunted the negative inotropic effect of ryanodine (SR calcium release channel regulator) (P < .01; ANOVA; n = 6). The positive inotropic effect of Bay K 8644 (sarcolemmal, L-type calcium channel regulator) was not affected by L-NMMA (P = NS; ANOVA; n = 6). L-NMMA had no effect on either [3H]ryanodine or [3H]PN200-110 (sarcolemmal, L-type calcium channel regulator) binding to cardiac membranes. These findings support a cGMP-dependent role for endogenous NO in myocardial E-C coupling.