Prostaglandins [PG] B2, E2 and F2.alpha. stimulated RNA synthesis in mammary gland explants of mice in a prolactin-like manner. These effects were non-additive to maximally stimulatory concentrations of prolactin. Time-course studies showed that the effects of these PG only became apparent following a 2-4 h lag period. The effects of these PG were not observed in explants which were not pretreated with insulin plus hydrocortisone, i.e., in tissues not responsive to prolactin. PG E1, A2, A1, B1 and F1.alpha. had no effects on the rate of RNA synthesis, but each of these PG abolished the responsivity of the explants to prolactin. Since dibutyryl cyclic AMP, theophylline and indomethacin were found previously to suppress the action of prolactin on RNA synthesis, the effects of these agents on the stimulatory effects of PG B2, E2 and F2.alpha. were tested. The incubation of explants with 5 mM dibutyryl cyclic AMP or 5 mM theophylline abolished the effects of these PG on RNA synthesis. But incubation with indomethacin did not affect the responses to these PG. Prolactin-like effects of PG on casein synthesis were not observed. PG E1, A2, A1, B2, F1.alpha. and indomethacin did abolish the prolactin stimulation of casein synthesis. The preincubation of explants with PG B2, E2, F2.alpha. or arachidonic acid significantly shortened the time of onset of the prolactin stimulation of casein synthesis. The results are discussed with regard to a possible role of the PG as mediators for the actions of prolactin in the mammary gland.