There is increasing evidence that the opioid peptide precursor, proenkephalin A, and its products undergo extensive post-translational modification, in addition to the cleavage at dibasic amino acid sites. We have used an antiserum directed toward the C terminus of Met-enkephalin Arg6-Phe7 in a radioimmunoassay to monitor the purification to homogeneity of four peptide B variants from bovine adrenal medulla, using gel filtration, anion exchange chromatography, and reverse phase high performance liquid chromatography. Amino acid sequence analysis, together with immunochemical data, confirmed that each comprised the primary sequence, proenkephalin A-(209-239). In addition, three of the four variants were shown to be phosphorylated by alkaline phosphatase digestion, microphosphate analysis, and ethanethiol derivatization coupled with amino acid sequence analysis; these variants were shown to have 1, 2, or 3 phosphate groups per peptide chain, which corresponded to their increasing acidic nature. The phosphorylation sites were clustered together at positions Ser7, Ser13, and Ser15 and were in close association with acidic residues. The clustering of phosphorylated residues is unique among regulatory peptide precursors. This region of proenkephalin A is well conserved, which suggests that it constitutes an important novel functional domain.