PRL has been reported to exist as a number of mol wt and charge variants. We have attempted to determine the relationships among the charge and lower mol wt variants by two-dimensional gel analysis of pituitary and pituitary cell extracts. Silver-stained gels of the extracts showed three major charge isoforms of 24,000 mol wt PRL (PRLs 1, 2, and 3, with PRL 3 being the most anodic) and what appeared to be an arc of products originating in the region of PRL 3. Each spot in the arc was distinct and represented a small decrease in size (two to eight residues), with a corresponding increase in net negative charge. When primary cell cultures were labeled with [35S] methionine, the three PRL isoforms and arc products were detectable by autoradiography. Western blots of the two-dimensional gels snowed the arc products to be immunologically related to PRL. Treatment of the cell cultures with hydroxynorvaline (5 mM), which inhibits processing of pre-PRL to PRL, resulted in doublet spots in the arc. Elimination of protease inhibitors or an increase in temperature during protein isolation had no effect on the relative concentrations of PRL and arc products. Inclusion of standard PRL or [125I]iodo-PRL in the extraction solution did not increase the size of the spots in the arc or produce labeled arc products, respectively. Treatment of the cell cultures with chloroquine (10–5 M) before and during radiolabeling had no effect on the production of radiolabeled arc products. Analysis of cell culture medium showed at least some of the arc products to be secreted. We conclude that PRL or a PRL-like molecule is processed intracellularly into a number of smaller derivatives. As the arc products accumulate within the cell and are secreted, we suggest that they may be biologically important relatives of PRL.