We observed in the HIT cell, a clonal insulin-secreting cell line, that epinephrine and somatostatin lower insulin mRNA levels and intracellular insulin content in addition to the well-recognized effect of these hormones to inhibit insulin secretion. To determine whether these inhibitory hormones might regulate insulin synthesis at the level of insulin gene transcription, we studied HIT cell expression of a human insulin-chloramphenicol acetyl transferase (CAT) reporter gene in the presence of glucose, epinephrine, and somatostatin. HIT cell expression of this human insulin-CAT reporter gene was responsive to glucose in a concentration-dependent manner, increasing threefold as the glucose concentration increased from 0.4 to 11 mM. Epinephrine significantly inhibited insulin-CAT reporter gene expression (61 ± 5% of control), an effect mediated specifically by the human insulin gene promoter/enhancer sequence. Somatostatin significantly inhibited expression of the human insulin-CAT reporter gene (65 ± 4% of control) and, to a lesser extent, expression of a control reporter gene, pRSVCAT (78 ± 4% of control). Thus, somatostatin may inhibit insulin gene transcription by insulin gene-specific effects as well as more general effects on gene expression. Both epinephrine and somatostatin inhibited expression of the human insulin-CAT reporter gene in a concentration-dependent manner that paralleled inhibition of insulin secretion. These studies indicate that epinephrine and somatostatin lower HIT cell insulin mRNA levels in part by inhibiting insulin gene transcription. Thus, hormonal inhibition of insulin secretion may be coupled with inhibition of insulin synthesis, thereby allowing the α-cell to match insulin supply to secretory demand.