This study compares the processing of pro-opiomelanocortin (POMC) at two Lys-Lys cleavage sites, located in the carboxy-terminal domain of the precursor, one site marking the amino terminus of beta-melanocyte-stimulating hormone (beta-MSH) and the other in the carboxy-terminus of beta-endorphin (beta E). These comparisons were carried out by transfecting monkey POMC cDNA into two heterologous cell lines: AtT-20, which endogenously expresses mouse POMC, and Rin m5F, which has been previously used as a host for transfected POMC. These cells lines are known to process POMC differently at Lys-Arg residues, though less is known about their Lys-Lys cleavage. Our results have demonstrated both tissue-specific and site-specific factors controlling Lys-Lys cleavage. The AtT-20 line appears not to perform either Lys-Lys cleavage. Rin m5F cells, on the other hand, fail to process the site at the carboxy terminus of beta E (beta E28-29) but do process, to a significant extent, the N-terminal site to beta-MSH. That this differential processing is unlikely to be due to a POMC conformation which would make the beta E site inaccessible was demonstrated by mutating the sites from Lys-Lys to Lys-Arg. With such mutants, Rin m5F cells fully processed at both locations. Interestingly, the mutant Lys-Arg sites were not fully processed by AtT-20 cells. These results are discussed in terms of the complement of processing enzymes expressed in each of the cell lines, as well as the role of residues surrounding the diabasic cleavage sites in determining the likelihood of proteolysis.