PRL-secreting epithelial cell lines (PECL) derived from Furth's MtT/W15 transplantable rat pituitary tumor have been studied as monolayer cultures for up to 18 months. Tumor epithelial cells were mechanically dispersed, plated at high density, and selectively enriched by picking and pooling colonies, thereby avoiding single cell-cloning conditions, in order to study the long term natural evolution of tumor cell populations in vitro. Fibroblast-like cells, prevalent in primary and early passages, underwent a series of morphological changes, which have been interpreted by others as in vitro aging or differentiation, and were eliminated from the cultures within 9 months. PECL phenotypes were heterogeneous and differed from sc MtT/W15 tumors with respect to morphological differentiation and modal chromosome number (48 for solid tumors: 48-92 for PECL; 42 for diploid). PECL were devoid of secretory granules by electron microscopy, but accumulated neutral lipid, complex lysosomes, and endocytotic vesicles. PECL differed from one another in plating efficiency (0.6-7.3%) and basal secretion rates for PRL and GH. Population doubling times decreased during the culture interval from 100 to 24-37 h in different PECL. RIA of medium from early cultures (less than 7 months) disclosed that PRL always exceeded GH levels, mimicking the serum hormone ratio of MtT/W15 tumor-bearing Wistar-Furth rats. Basal hormone release declined with time from a maximal initial rate of 93 micrograms/mg cell protein.24 h for PRL and 2.3 micrograms/mg cell protein.24 h for GH. GH production ceased by 7 months in all PECL, but PRL secretion persisted for 18 months. PRL production increased during exponential growth as a direct function of cell number and protein, and reached a maximum during confluency. Immunoperoxidase-conjugated antibody staining localized PRL in the Golgi region but not in secretory granules, consistent with a rapid release rate for the hormone. In preliminary experiments, rat hypothalamic extract (one hypothalamic extract RP-1 equivalent per dish) and 17 beta-estradiol (1 X 10(-8) M), but not TRH (5 X 10(-8) M), stimulated PRL secretion from PECL/F3, whereas only hypothalamic extract stimulated total cell protein after exposure to the agents for 3-6 days. Long term cultures of PECL exhibited reduced oncogenicity when transplanted in W/Fu rats. During serial passages, in vivo, PRL secretion decreased from 154 microgram/ml serum to the normal range within the same time framework that PRL secretion from cultured PECL/F3 was observed to decrease and stop in vitro. We believe that this phenomenon represents an expressed program within the cells rather than being merely a reflection of the in vitro environment.