In the presence of ATP and the absence of Ca2+, the binding of myosin subfragment-1 to actin is only slightly inhibited by troponin-tropomyosin, while the actin-activated subfragment-1 ATPase rate is 95% inhibited (Chalovich, J. M., Chock, P. B., and Eisenberg, E. (1981) J. Biol. Chem. 256, 575-578). On the other hand, it has been reported the troponin-tropomyosin markedly inhibits the binding of heavy meromyosin (HMM) to actin in the presence of ATP and the absence of Ca2+, providing that the HMM has intact light chain 2 (Wagner, P. D., and Stone, D. (1982) Biochemistry 22, 1334-1342). In the present study, we reinvestigated the binding of HMM with 85% intact light chain 2, to regulated actin. If we assume that only a single population of HMM is present, the binding constant of HMM to regulated actin at 19 mM ionic strength is only about 3 times larger in the presence of Ca2+ than in the absence of Ca2+ (2.4 X 10(4) M-1 compared to 8.8 X 10(3) M-1). On the other hand, if we correct for the population of HMM with degraded light chain 2, the difference in the binding constants in the presence and absence of Ca2+ may be as great as 5-fold. A double binding experiment also suggested that HMM with intact light chain 2 binds at most 5 times more strongly to regulated actin in the presence of Ca2+ than in its absence. We conclude that, just as with subfragment-1, the primary effect of troponin-tropomyosin in regulating the acto HMM ATPase activity is to inhibit a kinetic step in the ATPase cycle. However, our data with HMM also suggest that, in addition to this primary effect, troponin-tropomyosin may modulate the binding of the cross-bridge to actin in relaxed muscle to a small extent.