1. Fast skeletal myosin subfragment 1 (S1) was separated into two isozymes, S1(A1) and S1(A2), based on the associated alkali light chain, and their thermostabilities were compared. 2. Inactivation rate constants of Ca2(+)-ATPase (at 30 and 35 degrees C) were higher and heat-induced turbidity increase at 340 nm (at 40 degrees C) was faster with S1(A1) than with S1(A2), indicating a higher stability of S1(A2). 3. When S1 isozymes were incubated in the presence of excess alkali light chain, turbidity increase was markedly reduced, depending on the amount of light chain added. 4. Results obtained strongly suggest that alkali light chains are involved in the maintenance of myosin head structure.