Reversed-phase liquid chromatography has been used to extract and purify to homogeneity the two major forms of corticotropin (ACTH) from 60 rat anterior pituitaries. Tissue was homogenized in a medium designed to minimize peptidase activity and maximize solubilization of peptides. The supernatant obtained from the tissue homogenate was extracted in a batch procedure with octadecylsilylsilica (ODSsilica). Elution of the ODS-silica gave rise to a desalted and essentially protein-free preparation, which was enriched in peptides. From this initial extract, the two major forms of rat ACTH were purified to apparent homogeneity by reversedphase high-performance liquid chromatography (RP-HPLC), using solvent systems containing either trifluoroacetic acid or heptafluorobutyric acid as hydrophobic counterions. The recovery of ACTH immunoreactivity through the tissue ex-traction and chromatography stages was close to 100%. In control experiments, it was observed that the structural integrity of synthetic tritiated human ACTH was maintained throughout the extraction and purification procedures. The two forms of rat ACTH were found in approximately equimolar amounts, with very similar amino acid compositions which indicated a close similarity to other mammalian corticotropins. Both forms were found to have biological activities and molecular weights comparable to standard synthetic human ACTH1-39. Trypsin digestion indicated that the two peptides were identical except for a modification of one form in the carboxyl-terminal tryptic peptide. Initial radiolabeling experiments, using cultured rat anterior pituitary cells, have shown that the more polar form of rat ACTH is Ophosphorylated on the serine residue at position 31.