Two myosin heavy chain isoforms expressed in smooth muscle, SM1 (204 kDa) and SM2 (200 kDa), are derived from alternate splicing that results in different amino acid sequences at their non-helical C-terminal tail regions. These isoforms are developmentally regulated and differentially expressed in various smooth muscle tissues. The functional role of myosin isoforms differing at the C-terminal tail has been investigated both in vitro and in vivo. Removal of the C-terminal tail of SM1 by chymotrypsin activates the ATPase of myosin at low Mg2+ but does not change the maximum activity. Addition of peptides, mimicking C-terminal tail regions specific to the SM1 and SM2 isoforms, to permeabilized taenia coli smooth muscle fibers inhibits maximum shortening velocity (Vm) and decreases Ca2+ sensitivity but has no effect on maximum force. The inhibition of Vm by the SM1-peptide was not reversed on washout, whereas Vm inhibition by the SM2-peptide is reversible. We demonstrated that the SM1 peptide specifically bound to myosin at the subfragment 2-light meromyosin (S2-LMM) junction using crosslinking and immunomicroscopy. Modification at this site could have a direct effect on crossbridge function. The relation between C-terminal myosin isoforms and contractile function in vivo was examined using estrogen administration to ovariectomized rats to increase the relative expression of the SM1 C-terminal isoform in uterine smooth muscle. This increase in SM1 was significantly correlated with an increase in Vm. In contrast, the high ATPase N-terminal isoform was decreased by administration of estrogen to ovariectomized rats. Thus, changes in C-terminal isoform distribution appear to affect contractile function in vivo. We propose a mechanism whereby the interactions between the C-terminal tail of one myosin molecule and the S2-LMM region of another in the thick filament can modulate contractility in an isoform specific manner. Further work is needed to unequivocally identify the function of smooth muscle myosin isoforms. However, our evidence suggests that the C-terminal heavy chain isoforms may be important modulators of smooth muscle contractility.