We have isolated a cDNA clone for the human slow-twitch muscle isoform myosin light-chain 1slow-a (MLC1sa) from a skeletal muscle library and for the human nonmuscle isoform myosin light-chain 3nonmuscle (MLC3nm) from a fibroblast library. The nucleotide sequence of both isoforms was determined, and isoform-specific probes were constructed. In addition, MLC1sa was subsequently isolated from the fibroblast library. MLC1sa and MLC3nm were found to be very closely related to each other and distant from all other myosin light-chain isoforms so far described. We concluded that MLC1sa arose by duplication of MLC3nm rather than from any other isoform. A comparison was made between all human myosin light chains described to date and a model proposed for the evolution of this multigene family. A comparison between human and chicken myosin light-chain isoforms showed that human isoforms are more similar to their chicken counterparts than to human MLC1sa. The expression of MLC1sa and MLC3nm was studied in humans, rabbits, mice, and rats. MLC1sa was detected at the onset of both human and murine myogenesis in vitro. With development, MLC1sa may be replaced by the other slow-twitch muscle isoform, 1sb, in slow-twitch skeletal muscle, but the proportion of MLC1sa to 1sb expression varies between different species. MLC1sa was detected in nonmuscle cells in humans, mice, and rats. MLC3nm was the major nonmuscle alkaline myosin light chain in all species tested, but its pattern of expression in nonmuscle tissues was not identical to that of beta- or gamma-actin. We have shown that in the human, as in the chicken, one exon is spliced out of the MLC3nm transcript in smooth muscle to give an alternative product. We concluded that all alkali myosin light-chain isoforms may be functionally different.