A polypeptide containing approximately equal to 112 amino acid residues, with the thymosin alpha 1 sequence at its NH2 terminus, has been isolated from rat thymus by using a radioimmunoassay with an antibody prepared against synthetic thymosin alpha 1. The new polypeptide, named "prothymosin alpha," was found to be the major substance crossreacting with thymosin alpha 1 antiserum in rat thymus extracts; peptides corresponding to thymosin alpha 1 or thymosin alpha 11 were not detected. In gel filtration at pH 2.8, prothymosin alpha emerged as a single symmetrical peak corresponding to an apparent molecular weight of 32,000, approximately 3 times larger than the minimum molecular weight calculated from its amino acid composition. On the same gel filtration columns, synthetic thymosin alpha 1 (calculated Mr = 3108) emerged at a position corresponding to a molecular weight of 10,000-11,000. Thus, both prothymosin alpha and thymosin alpha 1 appear to exist in solution as oligomers, possibly as trimers. Prothymosin alpha and synthetic thymosin alpha 1 also were separated readily in reverse-phase HPLC and in isoelectric focusing; the isoelectric point of prothymosin alpha determined by the latter procedure was found to be 3.55, consistent with an unusually high content of glutamic and aspartic acids based on amino acid analyses. Prothymosin alpha appears to represent the native polypeptide from which thymosin alpha 1 and other fragments are generated during the isolation of thymosin fraction 5.