Abstract: The contribution of the prohormone‐processing enzyme PC2 to the proteolytic maturation of proenkephalin was examined in three sets of studies. In the first, the processing of this precursor was compared in PC2‐rich (Rin5f) and PC2‐lacking (AtT‐20) cell lines expressing proenkephalin by virtue of stable transfection. These studies showed that the time frame for processing of this precursor is cell line specific, with AtT‐20 cells processing proenkephalin to peptide B much more rapidly than Rin cells. However, the latter cell line processed proenkephalin much more extensively, i.e., produced a greater proportion of the penta‐ to octapeptide enkephalins. The involvement of PC2 in these later processing events was analyzed by examining the processing of proenkephalin in PC2‐overexpressing AtT‐20 cell lines. These experiments yielded a processing profile similar to that observed for Rin cells, although the time frame of initial processing was similar to that found in AtT‐20 cells. To confirm the physiological involvement of proenkephalin in the production of the small opioid peptides, we generated a Rin cell line in which the production of PC2 was impaired due to stable expression of antisense mRNA to this enzyme. These experiments provided conclusive evidence that the generation of Met‐enkephalin‐Arg‐Phe and Met‐enkephalin‐Arg‐Gly‐Leu, but not the larger enkephalin‐containing peptides, is mediated by PC2. Taken together, our data support the idea that PC2 is physiologically capable of mediating only the later processing steps of neuropeptide precursors. PC2 thus appears to be the primary enzyme responsible for the generation of bioactive opioid peptide species from proenkephalin.