The actin-activated ATPase activity of myosin subfragment 1 (S-1) isoenzymes, separated on the basis of their alkali light chain (A1 and A2) content, has been analysed under a number of different conditions. Previous experiments on the effects of increasing actin concentrations on the ATPase have shown that the maximum turnover rate of ATP by S-1 (A2) was about twice that of S-1 (Al), and the K , for actin was also considerably larger for this isoenzyme. Here we show that these kinetic differences are maintained when regulated actin (actin + tropomyosin + troponin) is used in place of purified F-actin. However, while increasing the ionic strength of the solution from 6 mM to 46 mM KCl has little effect on V for S-1 (A2), the corresponding value for S-1 (Al) increases to approximately equal that of S-1 (A2), suggesting that the two isoenzymes are controlled by the same rate-limiting process in the steady state under these conditions. The value of K , also increases with increasing ionic strength for the two isoenzymes, but that for S-1 (Al) increases more rapidly and approaches that for S-1 (A2). These experiments were carried out at at fixed S-1 concentration, but the maximum rate of actin activation of the S-1 ATPase can also be obtained from experiments where the actin concentration is fixed and the S-1 concentration varied. Under these conditions in 6 mM KCl, the maximum rate of ATP hydrolysis expressed per mole of actin at infinite S-1 concentration is the same for both s-1 (Al) and s-1 (A2), and this value numerically equals the maximum rate of hydrolysis of ATP by S-1 (A2) expressed at infinite actin concentration. These results suggest that the rate-limiting step in the ATPase cycle for S-1 (A2) is the release of products from an actin . S-1 . ADP . Pi complex, while for S-1 (Al), where the value of V at infinite actin concentration is considerably lower, a different process is rate limiting, and this process may occur before actin reassociation with the S-1 . ADP . Pi complex, using the Lymn-Taylor kinetic model. Thus increasing the ionic strength appears to change the rate-limiting process in the steady-state hydrolysis of ATP by actoS-1 (Al). These experiments are discussed in terms of the role of the alkali light chains in the myosin ATPase and muscle contraction.