Estrogens are known to exert specific stimulatory effects on basal and dopamine-inhibited PRL secretion and synthesis as well as on PRL gene expression. However, dihydrotestosterone (DHT) and progesterone (P), although inactive alone, can reverse the effect of 17 beta-estradiol (E2) on PRL release both in vivo and in vitro. Using castrated male rats, we have studied the effect of E2 (0.25 micrograms), P (2 mg), or DHT (100 micrograms) administered twice daily for 14 days alone or in combination on pituitary PRL mRNA levels measured by quantitative in situ hybridization. Treatment with E2 increased the accumulation of PRL mRNA by about 2.6-fold. Administration of P or DHT alone failed to modify PRL mRNA concentrations. However, DHT could prevent by 80% the stimulatory effect of E2 on PRL mRNA levels. Similar results were obtained by dot blot hybridization assay. The effects of sex steroids on PRL mRNA were closely paralleled to pituitary PRL content measured by RIA. The present data demonstrate that the effect of sex steroids on immunodetectable PRL result from a modulation of PRL mRNA accumulation. The sexual dimorphism observed in pituitary PRL content results from a 3.5-fold greater accumulation of PRL mRNA in intact females than in male rats. These results also clearly show that quantitative in situ hybridization is a powerful tool in the investigation of the regulation of gene expression in addition to providing valuable information on the localization of specific mRNA.