Effects of regulatory proteins on the actomyosin subfragment 1 (acto-S1) Mg2+-ATPase activity were studied over a range of S1 concentration at low actin concentration such that the specific activity was constant in the absence of the regulatory proteins. In their presence, the activity was inhibited at low [S1] and activated at higher [S1] in each of three cases: 1) troponin + tropomyosin + Ca2+; 2) tropomyosin; 3) troponin + tropomyosin - Ca2+. The [S1] required to activate increased in the order 1, 2, 3. In all three cases, however, the inhibition increased toward 100% when the [S1] approached zero. Tropomyosin titrations of acto-S1 ATPase at low [S1] which resulted in inhibition were hyperbolic and gave a binding constant of K approximately 10(7) M-1. In contrast, at higher [S1], tropomyosin titrations were sigmoidal, indicating cooperative effects on activation. These results suggest a modification of the simple steric blocking theory of regulation, in which it is postulated that both of the Ca2+-dependent positions of tropomyosin on the thin filament block the formation of active acto-S1-nucleotide intermediates at low [S1], and in which tropomyosin occupies a third "nonblocking" position in the active state at high [S1]. In this modified model, Ca2+ facilitates the binding of myosin heads, leading to the active site.