Clones for human prothymosin alpha have been identified in cDNA libraries from staphylococcal enterotoxin A-stimulated normal human lymphocytes and from simian virus 40-transformed fibroblasts. The 1198-base-pair fibroblast clone has been sequenced. The encoded protein is highly acidic (54 residues out of 111) and shares greater than 90% sequence homology with rat prothymosin alpha. The peptide "hormone" thymosin alpha 1 appears at positions 2-29 of the prothymosin alpha amino acid sequence. There is no N-terminal signal peptide. Examination of mouse and human tissues revealed the presence of prothymosin alpha mRNA in kidney, liver, spleen, normal lymphocytes (predominantly T cells), human T-cell leukemia virus-infected T cells, and myeloma cells (B-cell lineage). Prothymosin alpha mRNA is inducible; upon mitogen stimulation it increased greater than 15-fold above the level found in resting lymphocytes. Similarly, serum-deprived NIH 3T3 cells responded to serum restitution with an increase in prothymosin alpha mRNA. Characterization of human genomic DNA by Southern blot analysis disclosed a complicated pattern consistent with genetic polymorphism. These data suggest that prothymosin alpha plays an intracellular role tied to cell proliferation. There is no evidence that it serves as a precursor for secreted thymic peptides. However, given the complexity at the genomic level, multiple functions, including a putative secretory capability, cannot be excluded.