For isolated rat hepatocytes, glucagon, 3′:5′-cyclic AMP, 3′:5′-cyclic GMP, and epinephrine stimulate the rate of gluconeogenesis from substrates not involving pathways of mitochondrial metabolism. From estimation of the rates of glucose formation, fructose 6-phosphate phosphorylation, and lactate and pyruvate formation it is concluded that epinephrine and 3′:5′-cyclic GMP stimulate gluconeogenesis from either galactose or fructose by influencing the rate of reactions involving fructose 6-phosphate in a manner similar to that already reported for glucagon and 3′:5′-cyclic AMP. Each agent acts to inhibit flux through phosphofructokinase (EC 188.8.131.52) and enhance flux through fructose diphosphatase (EC 184.108.40.206), resulting in the re-direction of carbon from lactate and pyruvate formation to glucose synthesis. In addition to 3′:5′-cyclic GMP, dibutyryl 3′:5′-cyclic GMP, 8-bromo 3′:5′-cyclic GMP, 8-benzyl-thio 3′:5′-cyclic GMP and 8-(4-chlorophenyl)thio 3′:5′-cyclic GMP stimulate glucose formation and inhibit lactate and pyruvate formation from galactose. Guanosine monophosphate and 2′:3′-cyclic GMP are inactive. As the stimulatory effect of epinephrine is inhibited by phenoxybenzamine and not by propranolol, and is not simulated by isoproterenol, it is concluded that catecholamine activity is expressed through the α-receptor. Increased extracellular glucose concentration (>10 mM) decreases the stimulatory effect of epinephrine, 3′:5′-cyclic GMP, and partially that of 3′:5′-cyclic AMP but does not alter the efficacy of glucagon.