Thymosin beta 4 antibodies have been raised in rabbits by conjugating the peptide to keyhole limpet hemocyanin, and a radioimmunoassay for thymosin beta 4 has been established utilizing tritiated thymosin beta 4 as binding ligand. RIA values for thymosin beta 4 in rat tissues reveal levels ranging between 8 and 448 micrograms/g wet weight. Highest levels are observed in spleen, although the peptide is also relatively abundant in thymus, lung, and nasal epithelium. Brain, kidney, liver and testis contain only 10 to 20% the concentration of thymosin beta 4 seen in spleen, whereas heart and muscle contain only 5 to 10% that of spleen. However, thymosin beta 4 probably has a ubiquitous distribution in the rat, being present in all tissues examined. The specificity of the antiserum used in the RIA has been defined. The thymosin beta 4 molecule seemingly bears two major epitopes that lie between amino acid residues 1-8 and 22-32. The antiserum does not cross-react with other unrelated peptides. The antiserum will precipitate biosynthetically labeled beta 4 from cultured rat peritoneal macrophage.