Myosin subfragment 1 (S1) heavy chains were prepared from chicken muscle S1 by immunoadsorption in NH4Cl and from rabbit muscle S1 by ion exchange chromatography at 37 degrees C in MgATP. Both heavy chain preparations contained some intact S1, and the ATPase activities of both preparations were less than those of control S1. Recombinations of these heavy chains with alkali light chains prior to removing NH4Cl or MgATP lessened the decreases in ATPase activities. However, once NH4Cl or MgATP was removed, alkali light chain recombination did not result in any increase in ATPase activity. Thus, the lower activities appear to result from the instability of the S1 heavy chain in the absence of alkali light chain. When incubated with a 10-fold molar excess of radiolabeled alkali light chains for 1 h under approximately physiological conditions, almost all of the alkali light chains of S1 but only 8% of those of myosin or myofibrils were exchanged. Aggregation of myosin into filaments accounts for part of this difference in exchangeability. Removal of 30% of the 19,000-Da 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) light chains from myosin increased 3-fold the amount of alkali light chain exchanged. The exchange of the alkali light chains of S1 is inhibited by the presence of DTNB light chains, and cleavage of the DTNB light chains of heavy meromyosin to 17,000-Da fragments increased the rate of alkali light chain exchange. There was no detectable difference in the exchangeability of the two different alkali light chains.