PRL exists within the mammotroph population in a number of different molecular forms. Three of these forms are best described as isoforms, as they have the same mol wt (24K) but differ in their net molecular charges. In this study we have examined the relative proportions of newly synthesized isoforms found in stored (intracellular) vs secreted (extracellular) PRL. Dissociated cells from female rat anterior pituitaries were cultured for 48 h and then incubated in [35S]methionine (6 h; 37 C). Intracellular and medium proteins were then extracted and resolved by one- and two-dimensional polyacrylamide gel electrophoresis, followed by silver staining or autoradiography. Control experiments, in which biosynthetically labeled PRL was reextracted, ensured that the isolation conditions did not in themselves promote isoform interconversion. The relative proportions of the PRL isoforms were determined by densitometric scanning of developed autoradiograms. In the cell extracts, the relative proportions were 13.6 ± 2.1% isoform 1 (least negatively charged), 71.5 ± 3.26% isoform 2, and 14.7 ± 1.9% isoform 3 (most negatively charged). In the medium, the relative proportions were 60 ± 2.89% isoform 1, 20 ± 1.75% isoform 2, and 11 ± 1.14% isoform 3. When the labeling was performed in the presence of 0.5 mM cysteamine (an agent we show to distinguish between newly synthesized and older stored hormone and, hence, between the previously described functional subpopulations of mammotrophs), the same ratios of newly synthesized isoforms were secreted from the cells. We conclude that secretion of the different isoforms is more complex than simple proportional release of each form, and based on the cysteamine results, this nonproportional release cannot be attributed to release of one isoform per functional subpopulation of mammotrophs.