Isolated parenchymal hepatocytes prepared from fasted (24 hours) rats convert added dihydroxyacetone and xylitol to glucose. Monobutyryl cyclic adenosine-3',5'-mono-phosphate (mb-cAMP) stimulated the rate from dihydroxyacetone (plus 32%; N equals 28) but not from xylitol (plus 4%; N equals 27). Iodoacetate (0.15 to 0.20 mM) was an effective inhibitor of gluconeogenesis from lactate-pyruvate mixtures (72% inhibition); quinolinate (2.5 to 3.0 mM) was relatively ineffective (26% inhibition). Measurements of glucogenic intermediates formed from added dihydroxyacetone in hepatocytes (20% cell suspension) preincubated with iodoacetate provided evidence that phosphoglyceraldehyde dehydrogenase was inhibited. Inhibition of additional glycolytic-gluconeogenic enzyme(s) involved in dihydroxyacetone metabolism was not disclosed by the metabolite concentration data. Because iodoacetate partially inhibited gluconeogenesis from dihydroxyacetone in 5% cell suspensions but did not prevent stimulation of gluconeogenesis by mb-cAMP (plus 31%), additional inhibition, probably nonspecific, occurred. With dihydroxyacetone as substrate, mb-cAMP had only a slight effect on the concentration of fructose diphosphate (decrease) relative to control experiments (no mb-cAMP), but preinculation of the cells with iodoacetate made the mb-cAMP-induced decrease much greater. mb-cAMP caused a 47% decrease (SE, 8;N equals 9) in the assayable activity of phosphofructokinase in liver cells incubated with dihydroxyacetone but did not alter fructose diphosphatase activity significantly. Cyclic GMP and cIMP were shown to stimulate gluconeogenesis from dihydroxyacetone; both nucleotides also cause a decrease in the assayable activity of phosphofructokinase. With xylitol as substrate, mb-cAMP did not cause stimulation of gluconeogenesis, decrease in fructose diphosphate concentration, or decrease in phosphofructokinase activity. Our results indicate that phosphofructokinase might be an important controlling enzyme in gluconeogenesis and subject to regulation by cAMP.