Calponin, an actin/calmodulin-binding protein present in smooth muscle thin filaments, modulates the actin-myosin interaction and actomyosin ATPase activity of smooth muscle myosin II. Binding of myosin heads to actin under conditions that produce weak or strong binding induces conformational changes in actin. Polarized fluorimetric measurements of rhodamine-phalloidin complex and 1,5-IAEDANS specifically linked to actin in myosin-free muscle fibers (ghost fibers) and to Cys-707 in myosin head, respectively, revealed conformational changes, as determined from the changes in orientation and mobility of fluorescent probes, upon addition of calponin to ghost fibers. The effect of calponin on conformational changes produced upon binding of phosphorylated or dephosphorylated heavy meromyosin (HMM) was also determined. Subfragment-1 preparation modified with NEM (NEM-S1) or pPDM (pPDM-S1) were used as models of strong and weak binding, respectively. Calponin changed both the orientation of fluorophores on the actin and the flexibility of the actin filaments, as determined from the angle between an actin filament and the fiber axis. Changes in the flexibility of actin filaments and the orientation of fluorophores produced by phosphorylated smooth muscle HMM were similar to those seen with NEM-S1, which formed a strong-binding association with actin and caused the transition of actin monomers to the "on" state; calponin markedly inhibited this effect. In contrast, pPDM-S1 and dephosphorylated HMM induced weak binding and the transition of actin monomers to the "of" state, and these effects were enhanced by calponin. Furthermore, calponin decreased the velocity of actin filament movement over skeletal muscle myosin O gamma phosphorylated smooth muscle myosin heads in an in vitro motility assay. These results suggest that calponin induces modulation of smooth muscle contraction by inhibiting the force-producing (strong-binding) state of cross-bridges and involves changes in actin conformation.