In the presence of vanadate (Vi) and ADP, myosin ATPase forms a stable inactive complex (myosin.ADP.Vi) at the active site. To elucidate the nature of the inactive complex, we studied the effect of Vi plus ADP on the interaction of heavy meromyosin (HMM) with F-actin. 1) Viscosity measurements showed that the actin-HMM rigor complex was dissociated into actin and HMM by Vi and ADP (both 10(-3) M range). 2) When the HMM.ADP.Vi complex isolated by gel filtration was mixed with actin in the absence of free Vi, about 60% of the added HMM formed a complex with actin, and more than 70% of the HMM bound to actin released Vi and ADP. 3) When a mixture of the isolated HMM.ADP.Vi complex with actin was dialyzed against a buffer without free Vi and free ADP, only less than 10% of Vi and ADP, which were originally bound to the HMM, were retained in the dialysis tube after 4 days. In contrast, if actin was omitted, about 80% of Vi and ADP were retained. 4) These results indicate that the HMM.ADP-Vi complex is dissociated from actin, and that Vi and ADP originally trapped at the HMM active site can be almost completely released from the active site by actin if free (released) Vi and ADP are concomitantly removed.