A radioimmunoassay, using a rabbit antiserum directed against thymosin alpha 1, was employed to detect the presence of crossreacting peptides in rat tissues. Highest concentrations were present in thymus, but thymosin alpha 1 cross-reacting material was also detected in brain, liver, kidney, lung, and spleen, in amounts ranging from 15% to 65% of the quantities found in thymus. In each case, the major immunoreactive peptide, after extraction and purification by a procedure that avoids proteolytic modification, was identified as prothymosin alpha, a peptide containing approximately equal to 112 amino acid residues. Prothymosin alpha is believed to be the endogenous peptide from which thymosin alpha 1 and other fragments are formed by proteolytic modification during the preparation of thymosin fraction 5. No peptides corresponding in size and chromatographic behavior to thymosin alpha 1 were detected with the extraction procedure employed.