Control of lipogenesis by glucagon and cyclic nucleotide derivatives was examined in freshly isolated hepatocytes. [14C]Acetate was incorporated at a linear rate for 2 h into cellular lipids and for at least 6 h into medium lipids. About 80% of the incorporated label was recovered in fatty acids. Incorporation of [1-14C]acetate and 3H2O into cellular and medium lipids was inhibited by glucagon (K50 = 8 X 10(-10) M), dibutyryladenosine 3':5'-cyclic monophosphate (K50 = 7.5 X 10(-8) M), and guanosine 3':5'-cyclic monophosphate and its dibutyryl and 8-bromo derivatives, each with K50 = 2.9 X 10(-6) M. Glucagon (10 nM) reduced incorporation of [14C]acetate and 3H2O into fatty acid by 73 and 52%, respectively, and into cholesterol by 24 and 10%, respectively. When added together to hepatocytes at submaximally inhibitory concentrations, dibutyryl cAMP and dibutyryl cGMP exerted additive effects. However, maximal inhibitory concentrations of both produced the same effect as the addition of either nucleotide alone. Thus, this preparation of rat hepatocytes responded to physiological concentrations of glucagon and low concentrations of dibutyryl cAMP. Cyclic guanosine derivatives inhibited lipogenesis, but 100 times greater concentration was required when compared with dibutyryl cAMP. Dibutyryl cAMP and dibutyryl cGMP did not act synergistically.