Vanadate (Vi, 3-300 microM) reversibly inhibited force development elicited by micromolar Ca2+ in membrane-skinned fibers of smooth muscle from taenia coli and trachea of guinea pig. When relaxed fibers were preincubated with Vi, the contraction to Ca2+ was characterized by a peak response followed by a lower steady-state phase. The peak phase depended on the rate of contraction and the [Vi]and was absent after Vi incubation during a previous contraction. These observations were consistent with Vi binding to a site that was exposed during the cross-bridge cycle but absent in the relaxed state. The actin X myosin X ADP intermediate formed at the active site during the cross-bridge cycle is suggested as the site of action of Vi. A weak antagonism between Pi and Vi was demonstrated during contractions activated by myosin thiophosphorylation. High concentrations of Pi (6-12 mM) were needed to produce a small inhibition (10%) of maximal Ca2+-activated tension. Skinned fibers relaxed slowly after Ca2+ removal, and the absence of an active state suggested that tension was maintained by noncycling cross bridges. Both Vi and Pi increased the rate of tension loss by 10-fold, but Vi was 5-10 times more potent than Pi. It is suggested that Vi and Pi both act on the active site but that Pi has a more efficacious action on slowly cycling than rapidly cycling cross bridges.