To identify signals that direct the proteolytic processing and regulated secretion of human proenkephalin (hPE), we have transfected the hPE gene or minigene constructs into pituitary tumor cells, either rat GH4Cl cells or mouse AtT-20 cells. Cells transfected with either the hPE gene or minigene contained similar levels of methionine-enkephalin (ME)-containing peptides and hPE mRNA. In the GH4Cl clones, ME was present predominantly in high-molecular-mass forms (5-25 kDa). In contrast, the AtT-20 clones contained almost exclusively free ME and low-molecular-mass forms (< 5 kDa), with very little high-molecular-mass species present. Thus, among pituitary cells, corticotroph-derived cells appear better equipped to process hPE than lactotroph-derived cells. Despite limited proteolytic processing, GH4Cl clones secreted large amounts of unprocessed (> 20 kDa) hPE into the medium, making up to 10% of endogenous rat prolactin secretion. Both precursor and processed forms of ME were cosecreted acutely (< 1 h) with rat prolactin, and release of both polypeptides was stimulated up to 12-fold by secretagogues. Thus, complete proteolytic processing was not required for accurate targeting of hPE to the regulated secretory pathway. When transfected with constructs bearing deletions of amino-terminal amino acids 2-43 or 2-67, i.e., part or nearly all of the synenkephalin moiety, GH4Cl cells handled the modified protein much like cells expressing the complete protein. They did not process the modified hPE extensively, but the protein was correctly targeted to the regulated secretory pathway. AtT-20 cells transfected with truncated hPE cDNA constructs expressed and processed the protein as efficiently as cells expressing unmodified hPE and expressed predominantly low-molecular-mass forms of ME. Therefore, the structural features required for correct targeting and processing are not present in the cysteine-rich amino-terminal third of the prohormone. It is interesting that the deletions did not include the SHLL peptide motif in synenkephalin, a motif that has been proposed as a sorting signal.