The alkali 1-type isoforms of myosin essential light chains from vertebrate striated muscles have an additional 40 or so amino acids at their N terminus compared with the alkali 2-type. Consequently two light chain isoenzymes of myosin subfragment-1 can be isolated. Using synthesized peptide mimics of the N-terminal region of alkali 1-type essential light chains, we have found by 1H NMR that the major actin binding region occurred in the N- terminal four residues, APKK.... These results were confirmed by mutating this region of the human atrial essential light chain, resulting in altered actin-activated MgATPase kinetics when the recombinant light chains were hybridized into rabbit skeletal subfragment 1. Substitution of either Lys3 or Lys4 with Ala resulted in increased K(m) and k(cat) and decreased actin binding (as judged by chemical cross-linking). Replacement of Lys4 with Asp reduced actin binding and increased K(m) and k(cat) still further. Alteration of Ala1 to Val did not alter the kinetic parameters of the hybrid subfragment 1 or the essential light chain's ability to bind actin. Furthermore, we found a significant correlation between the apparent K(m) for actin and the k(cat) for MgATP turnover for each mutant hybrid, strengthening our belief that the binding of actin by alkali 1-type essential light chains results directly in modulation of the myosin motor.