We report the isolation, sequence, and identification of a cDNA encoding the human kinesin light-chain (KLC) protein. The cDNA molecule consisted of 276 nucleotides of 5' untranslated region, the complete coding sequence of 1,710 nucleotides, and 322 nucleotides of 3' untranslated region. It encoded a polypeptide of 569 amino acids and a deduced molecular mass of 64,789 daltons. The predicted secondary internal structure of the KLC molecule consisted of about 27 contiguous repeats, each of approximately 21 amino acid residues, and could be divided into three domains. The amino-terminal domain consisted of heptad repeats typical of the rod domain of several cytoskeletal proteins. The central and carboxy-terminal domains consist of 21-mer repeats. KLC mRNA was expressed in most tissues analyzed. The gene, which was expressed in bacteria and Chinese hamster ovary cells, was provisionally assigned to the long arm of human chromosome 14.