The sites of action for somatostatin and epinephrine to inhibit insulin secretion have been reported to be exclusively in the exocytotic pathway. We used HIT cells, a clonal line of β-cells, to examine whether these hormones might have as yet undescribed, nonexocytotic effects on insulin messenger RNA levels. We observed that both somatostatin and epinephrine not only inhibit insulin secretion (53 ± 2% and 50 ± 2% of control, respectively) but also decrease insulin mRNA levels (54 ± 5% and 66 ± 5% of control, respectively) and insulin content in HIT cells (61 ± 2% and 51 ± 1% of control, respectively). The latter two effects are discernible by 24 h, maximal by 48 h, and are prevented by preincubation of HIT cells with pertussis toxin. These new observations suggest that somatostatin and epinephrine negatively modulate insulin availability through a guanine nucleotide binding protein-mediated step in insulin synthesis before the exocytotic pathway. This general mechanism may allow these two hormones to serve as more long-term regulators of insulin availability in distinction to their shorter term and more readily reversible inhibitory effects on the exocytotic pathway.