A somatomammotroph cell line derived from male rat anterior pituitary (rPCO) has been established without the use of transforming agents and has been maintained in culture for more than 1 yr (45 passages) using Minimum Essential Medium supplemented with 10% horse serum, 5 nM T3, 2 nM corticosterone, and 0.1 nM GH-releasing hormone (GRH). Peroxidase and immunofluorescent staining revealed immunoreactive GH in 99% of rPCO cells and immunoreactive PRL in 72% of cells. Within individual cells, GH and PRL appear to be colocalized. The storage capacity for GH in rPCO cells represented 40% of the daily hormone production. In serum-free medium containing 5 nM cortisol, GH secretion was stimulated 10- and 25-fold by 50 pM and 50 nM T3, respectively. GRH (1 nM) stimulated GH secretion 8-fold in the absence of T3, although no effect was observed in the presence of 50 nM T3. Qualitatively similar changes occurred in GH mRNA responses to T3 and GRH. Other secretory proteins were detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of culture medium, several of which were present in concentrations similar to that of GH. Nine separate cell lines were cloned from rPCO cells by limiting dilution, all of which secreted GH and PRL. GH secretion varied 6-fold between clones, and the GH to PRL ratio varied more than 200-fold. These rPC cell lines provide a new model for studying the control of GH and PRL gene expression, including hormone synthesis, processing, and secretion. They may also be useful for identifying other pituitary secretory products and as a source for the production of protein hormones.