Rat proenkephalin was overexpressed in Chinese hamster ovary cells using the dihydrofolate reductase-coupled genetic amplification method. About 2 mg purified protein could be obtained from 250 ml conditioned medium; multiple successive harvests could be obtained from the same roller bottle. Degradation of proenkephalin released into the conditioned medium was reduced significantly in the presence of 2% fetal bovine serum. Forty-eight percent of recombinant proenkephalin was glycosylated; glycosylation could be entirely prevented by the addition of tunicamycin. Two-dimensional isoelectric focusing experiments showed that recombinant proenkephalin exhibited considerable charge heterogeneity, with two major unglycosylated isoelectric forms and six or seven glycosylated isoelectric forms. The estimated isoelectric points of the major unglycosylated proenkephalins were 6.0 and 6.1, while glycosylated proenkephalins ranged in pI from 5.7-6.1. Some of this isoelectric heterogeneity is due to phosphorylation; [32P] orthophosphate was readily incorporated into serine residues within newly synthesized proenkephalin.