The present studies were undertaken to investigate the effect of gender on thyrotropin-releasing hormone (TRH) gene expression in cultured anterior pituitary (AP) cells. AP cells derived from 15-day-old male, female, or female pups that had been neonatally treated with testosterone propionate (TP), were cultured for up to 18 days in a modified DMEM/L-15 medium containing 10% fetal calf serum. TRH and AP hormones including GH, prolactin (PRL), luteinizing hormone (LH) and thyrotropin (TSH) were measured by RIA, proTRH mRNA was determined by in situ hybridization using a full-length riboprobe followed by quantification with a computer-assisted image analysis system. Cultures derived from female rats contained significantly (p < 0.01) higher amounts of TRH and secreted approximately twice (p < 0.01) as much TRH under basal conditions and in response to activators of the protein kinase A and C pathways, respectively. In situ hybridization studies revealed that 'female' cultures contained significantly higher amounts of proTRH mRNA compared to 'male' cultures. Computer-assisted image analysis demonstrated that proTRH mRNA levels were 3.5 times higher in 'female' compared to 'male' cultures (p < 0.01), an effect that was the result of a significantly higher number (3 times; p < 0.01) of cells expressing proTRH mRNA in 'female' cultures. Neonatal TP treatment did not affect either proTRH mRNA or TRH peptide levels. In vitro testosterone treatment resulted in a moderate rise (p < 0.05) of intracellular TRH accumulation in cultures from both sexes, however, proTRH mRNA levels remained unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)