The biosynthesis and initial processing of the methionine-enkephalin precursor preproenkephalin A were examined by cell-free translation of mRNA from brain and adrenal medulla. A novel antiserum raised against Met-enkephalin-Arg6-Phe7 was shown to react with bovine adrenal medulla fractions (apparent Mr 34,000) containing proenkephalin A. Affinity-purified antibodies from this antiserum were used to immunoprecipitate cell-free translated [35S]Met-enkephalin-containing protein. A protein of apparent Mr 30,000 +/- 500 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was the only Met-enkephalin precursor consistently synthesized by translation of mRNA from bovine or guinea pig striatum, rat brain, or bovine adrenal medulla. The presence of [35S]Met-enkephalin sequences in this protein was confirmed by high pressure liquid chromatography of trypsin/carboxypeptidase B digests. Dog pancreas endoplasmic reticulum membranes converted the Mr 30,000 protein to an immunoreactive protein of apparent Mr 28,500 that lacked significant core glycosylation. These results suggest that 1) a protein similar or identical to bovine adrenal medullary preproenkephalin A is the major Met-enkephalin precursor synthesized in brain as well as adrenal medulla, and 2) preproenkephalin A is converted to a protein resembling proenkephalin A, presumably by removal of a signal peptide.