Skeletal myosin has two isoforms of the essential light chain (ELC), called LC1 and LC3, which differ only in their N-terminal amino acid sequence. The LC1 has 41 additional residues containing seven pairs of Ala-Pro, which form an elongated structure, and two pairs of lysines located near the N-terminus. When myosin subfragment-1 (S1) binds to actin, these lysines may interact with the C-terminus of actin and be responsible for the isoform specific properties of myosin. Here we employ cross-linking to identify the LC1 residues that are in contact with actin. S1 was reconstituted with various LC1 mutants and reacted with the zero-length cross-linker 1-ethyl-3-[3-dimethyl-aminopropyl]-carbodiimide (EDC). Cross-linking occurred only when actin was in molar excess over S1. Wild-type LC1 could be cross-linked through the terminal alpha-NH2 group, as well as via the two pairs of lysines. In a mutant ELC, where the lysines were deleted but two arginines were introduced near the N-terminus, the light chain could still be cross-linked via the terminal alpha-NH2 group. When the charge was reduced in the N-terminal region while retaining the Ala-Pro rich region, the mutant could not be cross-linked. These results suggest that as long as the N-terminus contains charged residues and an Ala-Pro rich extension, the binding between LC1 and actin can occur.