Carboxypeptidase H (CP-H, EC 18.104.22.168) removes C-terminal basic amino acids from insulin and many other peptide hormone intermediates in endocrine and neural tissues. CP-H was studied in two rat insulinoma-derived clonal beta-cell lines that were chosen in part because their profile of hormone production is restricted. Although RIN 1046-38 cells predominantly produce insulin and RIN 1046-44 cells contain no known polypeptide hormone, both cell lines contain comparable levels of CP-H. The distribution of CP-H in RIN cells differs from primary tissues since only 25% of cellular CP-H is soluble whereas approximately 75% is associated with the membrane fraction. Immunoblotting with both N- and C-terminal antibodies detects a single 54 kilodalton (kDa) band corresponding to the mature form of CP-H in soluble and membrane RIN cell extracts and incubation media. Pulse-chase analysis shows that a 56 kDa precursor is initially synthesized and rapidly processed to a mature 54 kDa form. Newly synthesized, mature CP-H is detectable in the media by 30 min. Approximately 50% is retained within cells after 3 h of incubation. Secretion of CP-H activity from both RIN cell types and insulin from 1046-38 cells is acutely stimulated by membrane depolarization with KCl and attenuated by 10 mM MgCl2. Dense core secretory vesicles characteristic of the regulated secretory pathway are evident on transmission electron microscopy of both RIN cell lines. Thus, mature CP-H exists primarily as a membrane-associated form in RIN cells, but all forms of CP-H, including soluble and secreted enzyme, retain an intact C-terminus.