The kinetics of actin-dependent MgATPase activity of skeletal muscle myosin subfragment 1 (S1) isoform containing the Al essential light chain differ from those of the S1 isoform containing the A2 essential light chain. The differences are due to the presence of the extra N-terminal peptide comprising 42 amino acid residues in the Al light chain. This peptide can interact with actin; heretofore, there have no been reports of the direct interaction between this peptide and the heavy chain of S1. Here, using the zero-length cross-linker 1-ethyl-3-(3-dimetylaminopropyl)carbodiimide (EDC) and S. aureus V8 protease, we show for the first time that the N-terminal part of the Al-light chain can interact with the 22-kDa fragment of the S1 heavy chain. No such interaction has been observed for the S1(A2) isoenzyme. Localization of residues which can possibly react with the cross-linker suggests that the interaction might involve the N-terminal residues of the Al light chain and the converter region of the heavy chain.