A cDNA clone encoding human fast skeletal myosin regulatory light chain (HSRLC) has been isolated and characterized from a fetal muscle cDNA library. The cDNA contains the coding sequence of 170 amino acids (aa) and 58 and 91 nucleotides in the 5' and 3' untranslated regions (UTRs), respectively. HSRLC is encoded by a single copy gene in the human genome and shows a tissue-specific pattern of expression in skeletal muscle. Comparison of derived amino acid sequence of HSRLC with database sequences reveals highly conserved 12 amino acid residues in a putative calcium-binding region. HSRLC is unique among all RLC sequences in having three consecutive potential phosphorylatable serine residues. The Cys-129 of HSRLC corresponds to the critical Gly-117 of scallop RLC that is essential for its regulatory function. The clusters of hydrophobic residues that are believed to stabilize the binding of NH2-terminal of RLC with myosin heavy chain show high sequence conservation in RLCs. Besides identifying specific targets for functional studies of HSRLC by mutagenesis, the results support the concept of an ancestral gene from which the RLC genes have evolved.