The rate of glucose utilization in isolated pancreatic islets of the rat was inhibited by the α2-adrenoceptor agonists clonidine and epinephrine. Yohimbine reversed the inhibition. α1 or β-adrenoceptor agonists had little or no effect on glucose utilization. Stimulation of muscarinic receptors by carbamylcholine reversed the effect of clonidine. Pertussis toxin blocked the effect of clonidine on glucose utilization, and potentiated the response to carbamylcholine. 8-Bromo-cAMP did not affect glucose utilization in the presence of clonidine. Thus, α2-adrenoceptors negatively modulate glucose utilization, and the effect is mediated by an inhibitory guanine nucleotide regulatory protein, but not by cAMP.