Three naturally occurring variants of myosin light chain 1, type I, II, and III from avian fast-twitch muscle, have been analyzed by reverse-phase HPLC peptide mapping and amino acid sequencing. Difference peptides were absent from accompanying digests of the related protein, myosin light chain 3, indicating that the heterogeneity was located in the N-terminal 50 residues unique to light chain 1. The type II variant possessed the previous published sequence for the protein [Nabeshima Y., Fujii-Kuriyama, Y., Muramatsu, M., & Ogata, K. (1984) Nature (London) 308, 333-338]. The type I variant, which migrates faster than the type II on SDS gene electrophoresis, contained a Pro----Ala substitution at residue 15, turning the Lys-Pro-(Ala)5(Pro-Ala)7 stretch in this region into Lys-Pro-(Ala)7(Pro-Ala)6. The type III variant, which migrates just faster than the type I, had an (Ala)2 deletion in the (Ala)5 run, yielding Lys-Pro-(Ala)3-(Pro-Ala)7. As indicated by the SDS gel migration rates, the type I and III variants are significantly shorter in length than the type II. The benign nature of the changes is consistent with a flexible arm function for the N-terminal region of light chain 1, with the structural changes in the variants occurring in the spacer region of the arm.(ABSTRACT TRUNCATED AT 250 WORDS)