We previously reported the cloning of a rat S1 cDNA whose deduced amino acid sequence shares high similarity (92%) with that of mammalian elongation factor-1 alpha (EF-1 alpha), a protein involved in the binding of aminoacyl-tRNA to the ribosome during peptide synthesis. We report here the isolation of a full-length cDNA from a mouse brain library and a partial-length cDNA from a human hippocampus library which share extensive sequence similarity to rat S1 cDNA. We show that, as with mammalian EF-1 alpha S, the predicted primary amino acid sequences of rat, mouse, and human S1 are almost identical, except for one conservative substitution. These results indicate that mouse and man contain a second member of the EF-1 alpha gene family, the S1 gene. They also suggest that our result obtained in rat may be extrapolated to mouse and man.