We have previously shown that insulin suppresses growth hormone (GH) messenger (m) RNA levels in rat pituitary cells. To further delineate the molecular mechanism of insulin action, the effect of insulin treatment on GH gene transcription rates was examined in GH3 pituitary cells grown in serum-free defined medium. A transcriptional run-off assay was performed when intact isolated nuclei were allowed to continue RNA synthesis in an in vitro reaction. Specific incorporation of [32P]GTP into RNA was quantified by hybridization to rat GH complementary (c) DNA. Hybridization efficiency was measured with an internal [3H]cRNA standard and ranged from 30 to 48%. Alpha-amanitin (1 microgram/ml) inhibited total transcription, and excess unlabeled rat pituitary mRNA (250 ng) competitively inhibited GH mRNA hybridization by greater than 80%. Insulin (0.7 nM) inhibited new GH mRNA synthesis, and maximal inhibition (30% of control) was observed with 7 nM insulin after 4 h treatment. The inhibitory effects of insulin on new GH mRNA synthesis were abolished by both insulin-receptor-antiserum and by guinea-pig anti-insulin serum. The results show that insulin exerts a rapid suppression of new GH mRNA synthesis. These data provide evidence for the direct transcriptional regulation of the GH gene by insulin.