The activity of acetyl-CoA carboxylase (ACC), the rate-limiting enzyme of fatty acid biosynthesis, can be regulated by both adenine and guanine nucleotides in vitro. We have employed two inhibitors of IMP dehydrogenase, ribavarin and tiazofurin, to investigate a possible role for intracellular nucleotides in ACC regulation in rat adipocytes. Ribavarin, but not tiazofurin, leads to a profound time-dependent inhibition of ACC activity that is associated with a decrease in both intracellular ATP and GTP. This inactivating effect is largely reversed with guanosine, accompanied by increases in both ATP and GTP levels. Epinephrine-mediated inactivation of ACC in intact cells is not altered by ribavarin incubation. However, in these experiments, insulin-mediated activation is observed only after ribavarin-induced inhibition of the enzyme. These data suggest that nucleotides may modulate ACC activity and influence is regulation by insulin in intact cells. The possible mechanisms underlying the insulin activation of ACC and the role of intracellular nucleotides in insulin action are discussed.