PRL compartments have been studied in normal rat pituitary cells cultured for 6 days. The cells were pulse-labeled for 15 min with 35S-methionine and then chased for 24 h in the absence or presence of cycloheximide (3.6 X 10(-5) M). TRH (30 nM) was introduced into the medium either at the beginning or after increasing durations of chase. The findings were compared with those obtained with GH3B6 cells in similar experimental conditions. Despite the fact that normal PRL cells differ from GH3B6 cells by a large intracellular PRL store, several similarities were found between the two systems: newly synthesized PRL was rapidly and preferentially released in basal conditions, the pattern of the decay of the specific radioactivity of PRL released into the medium suggested the existence of at least two PRL pools with different half-lives: 2.5 h and 22 h, respectively, TRH induced the preferential release of stored PRL synthesized before the pulse, only 20% of the pulse-labeled PRL was released into the medium after 24 h of chase. However, normal PRL cells differed in several respects from GH3B6 cells: the turnover time of the two PRL pools is 8 times greater in normal PRL cells, an asynchrony in the time of appearance of labeled PRL in the medium was observed, suggesting a functional heterogeneity of these cells, at the end of the chase, 40% of the pulse-labeled PRL was lost in the case of normal cells, but not of GH3B6 cells, and this was prevented by cycloheximide, polyacrylamide gel electrophoresis analysis of this labeled immunoprecipitated intracellular material revealed the existence, in addition to the mol wt of 23,000 PRL and the large PRL-like forms (mol wt, 45,000 and 50,000), as observed with GH3B6 cells, of smaller proteins (mol wts, 39,000, 36,000, 20,000, 18,000, 15,000), which might represent degradation products.