We have successfully prepared active subfragment-1 (S1) of an air-breathing teleost Channa gachua by digesting natural actomyosin (NAM) with chymotrypsin, instead of using myosin as the primary source. Conditions of preparation of acto-S1 were worked out following SDS-PAGE profiles of degradation of 200 kD MyHC to 130 kD and 94 kD products, corresponding respectively to myosin rod and S1, up to 1 hr of incubation with chymotrypsin at 15°C. The observed cleavage was accompanied by 250% activation of NAM-Ca2+-ATPase during early 30 min of digestion. Actin from acto-S1 (prepared by chymotrypsin digestion of NAM) was dissociated by Mg2+-ATP and ammonium sulfate fractionation. S1 of C. gachua prepared by this protocol had a heavy chain of 94 kD and a light chain of 19 kD. It possessed satisfactory levels of Ca2+ and K+- ATPase and Ka for dissociation of actin. The two most remarkable biochemical characteristics were : activity maxima at neutral pH and the exceptional thermostability of Ca2+ -ATPase, being the highest of any fish S1 reported so far.