The major physiological inhibitors of insulin secretion, norepinephrine, somatostatin, galanin and prostaglandin E₂, act via specific receptors that activate pertussis toxin (PTX)-sensitive G-proteins. Four inhibitory mechanisms are known. 1. Activation of K_ATP channels and repolarization of the cell, 2. Inhibition of L-type Ca²⁺ channels, 3. Decreased activity of adenylyl cyclase, and 4. Inhibition of exocytosis at a "distal" site in stimulus-secretion coupling. We have examined the underlying mechanisms of inhibition at this distal site. In rat pancreatic islets, 2-bromopalmitate, cerulenin and polyunsaturated fatty acids, all of which suppress protein acyl transferase activity, blocked the distal inhibitory effects of norepinephrine in a concentration-dependent manner. In contrast, control compounds such as palmitate, 16-OH palmitate and etomoxir, which do not block protein acylation, had no effect. Furthermore, 2-bromopalmitate also blocked the distal inhibitory actions of somatostatin, galanin and prostaglandin E₂. Importantly, neither 2-bromopalmitate nor cerulenin affected the action of norepinephrine to decrease cyclic AMP production. We also examined the effects of norepinephrine, 2-bromopalmitate and cerulenin on palmitate metabolism. Palmitate oxidation and its incorporation into lipids seemed not to contribute to the effects of 2-bromopalmitate and cerulenin on norepinephrine action. These data suggest that protein acylation mediates the distal inhibitory effect on insulin secretion. We propose that the inhibitors of insulin secretion, acting via PTX-sensitive G proteins, activate a specific protein acyl transferase causing the acylation of a protein or proteins critical to exocytosis. This particular acylation and subsequent disruption of the essential and precise interactions involved in core complex formation would block exocytosis.