Prothymosin alpha and thymosin alpha 1 are believed to be thymus-derived, hormone-like materials with immunomodulatory functions performed outside the cell. These functions are inconsistent with the existence of a full length cDNA clone that does not encode an amino-terminal signal peptide or several consecutive hydrophobic residues. A study of the prothymosin alpha mRNAs and genes was undertaken in search of evidence for secreted forms of the protein. Prothymosin alpha mRNA was localized exclusively on free, rather than membrane-bound, polysomes. Upon screening cosmid and plasmid libraries totaling 2 X 10(6) clones, a gene family consisting of six members was identified. Sequence information from the 5'-ends of all the genes indicated that none encodes an amino-terminal signal peptide. One of the genes, apparently by means of alternate splicing, gives rise to two prothymosin alpha mRNAs, one of which has an additional internal glutamic acid codon with respect to the other. Comparison of the translated nucleic acid sequences of the five remaining genes with those encoded in the mRNAs revealed 30-98% homology in the first 50 amino acids. These five genes appear to be processed genes and/or pseudogenes. The localization of prothymosin alpha mRNAs on free polysomes, together with the partial nucleotide sequences of the genes, strongly suggest an intracellular function for prothymosin alpha. Therefore, the possibility must be raised that prothymosin alpha and its peptide derivatives act as xenobiotics when introduced into assays of immune function.