Thyrotropin-releasing hormone (TRH) stimulates prolactin production in cultured GH3 rat anterior pituitary tumor cells. For correlation of cell-by-cell prolactin distribution and intracellular hormone concentration, GH3 cells were grown to plateau-phase density on glass coverslips in plastic dishes. Acetone-fixed, cell-bearing coverslips were stained for prolactin by an immunoglobulin-peroxidase bridge technique (Mason et al., '69); cells on the plastic dishes were assayed for prolactin (microcomplement fixation immunoassay, Tashjian, '73) and protein content. Intracellular prolactin, unaffected quantitatively by acetone fixation and choice of substratum, was localized immunocytochemically by a granular brown precipitate, abolished if anti-prolactin serum was preabsorbed with rat prolactin or omitted from the protocol. Intracellular prolactin was maximized with colchicine (5.0 X 10(-6) M; final 3 hr of incubation) in control and TRH-treated (10 ng/ml; 48 hr) GH3 cell cultures. A total of 8,500 cells were classified by light microscopy as unstained, heavily (H) or moderately (M) stained for prolactin. In controls, 35% of cells were prolactin-positive: 5% H and 29% M. After TRH, 45% were positive: 7% H and 38% M. Although prolactin-positive cells were unevenly distributed, comprising 25% to 46% of cells in individual microscopic fields in controls, TRH increased the proportion of M cells in all areas. TRH treatment raised prolactin levels to 450% of control, but mathematical analysis attributed less than 30% of the increase to new prolactin-positive cells. We conclude that TRH acts on GH3 cultures principally by raising the mean hormone content of individual positive cells rather than by increasing the proportion of cells committed to prolactin production.