Nitrovasodilators such as nitroglycerine, via production of nitric oxide and an increase in [cGMP], can induce arterial smooth muscle relaxation without proportional reduction in myosin light chain (MLC) phosphorylation or myoplasmic [Ca2+]. These findings suggest that regulatory systems, other than MLC phosphorylation and Ca2+, partially mediate nitroglycerine-induced relaxation. In swine carotid artery, we found that a membrane-permeant cGMP analogue induced relaxation without MLC dephosphorylation, suggesting that cGMP mediated the relaxation. Nitroglycerine-induced relaxation was associated with a reduction in O2 consumption, suggesting that the interaction between phosphorylated myosin and the thin filament was inhibited. Nitroglycerine-induced relaxation was associated with a 10-fold increase in the phosphorylation of a protein on Ser16. We identified this protein as heat shock protein 20 (HSP20), a member of a family of proteins known to bind to thin filaments. When homogenates of nitroglycerine-relaxed tissues were centrifuged at 6000 g, phosphorylated HSP20 preferentially sedimented in the pellet, suggesting that phosphorylation of HSP20 may increase its affinity for the thin filament. We noted that a domain of HSP20 is partially homologous to the 'minimum inhibitory sequence' of skeletal troponin I. The peptide HSP20110-121, which contains this domain, bound to actin-containing filaments only in the presence of tropomyosin, a characteristic of troponin I. High concentrations of HSP20110-121 abolished Ca2+-activated force in skinned swine carotid artery. HSP20110-121 also partially decreased actin-activated myosin S1 ATPase activity. These data suggest that cGMP-mediated phosphorylation of HSP20 on Ser16 may have a role in smooth muscle relaxation without MLC dephosphorylation. HSP20 contains an actin-binding sequence at amino acid residues 110-121 that inhibited force production in skinned carotid artery. We hypothesize that phosphorylation of HSP20 regulates force independent of MLC phosphorylation via binding of HSP20 to thin filaments and inhibition of cross-bridge cycling.