A nonrepetitive complementary RNA probe specific for an intervening sequence of the rat pro-opiomelanocortin (POMC) gene primary transcript was used to analyze the hormonal regulation of POMC gene transcription in individual cell nuclei in the rat pituitary by in situ hybridization. This probe recognized only full-length POMC heterogeneous nuclear RNA, as verified by Northern blots of pituitary RNA. When pituitary sections were hybridized with this 3H-labeled POMC intron A probe, silver grains were predominantly localized over the nuclei of cells that expressed POMC in the anterior and intermediate lobes. Adrenalectomy increased both the average grain density over corticotroph nuclei and the number of cells in the anterior pituitary with significant numbers of silver grains over their nucleus. Dexamethasone administration to intact or adrenalectomized rats results in the rapid (within 30 minutes) disappearance of silver grains over the nuclei of corticotrophs in the anterior lobe, suggesting that POMC gene transcription had been inhibited. However, adrenalectomy or dexamethasone administration did not alter the silver grain density over nuclei of intermediate lobe melanotrophs. Thus, this in situ hybridization assay utilizing an intervening sequence-specific POMC probe can measure rapid physiological changes in POMC heterogeneous nuclear RNA in individual cell nuclei.