In rat hepatocytes freshly isolated from donor rats at different times of the day, the rates of lipogenesis (de novo fatty acid synthesis) varied with a diurnal periodicity. The maximal rate occurred approximately 5 hr after the end of the normal 8-hr feeding period and at this time was four- to fivefold higher than the minimum rate which occurred midway through the feeding period. A similar diurnal pattern of change persisted even when the supply of lipogenic substrate, present in the medium as pyruvate, was not limiting. Although insulin stimulated the basal rates of lipogenesis to different relative extents in hepatocytes isolated at different times of the day, in absolute terms the hormone had little effect on the overall pattern of change during the diurnal cycle. The presence of pyruvate protected lipogenesis against inhibition by glucagon. The degree of protection varied over the diurnal cycle. During the early stages of starvation (up to 24 hr) there was a continuous decline in the rate of hepatocyte lipogenesis, irrespective of whether insulin and/or lipogenic substrate (pyruvate) were available or not. After this time the decline in the rate of lipogenesis was much less rapid. Seventeen hr after removal of food from donor rats, a point was reached beyond which pyruvate was incapable of supporting the maximum basal rate of lipogenesis which occurred during the normal diurnal cycle of fed rats. After this time lipogenesis in the presence of pyruvate was inhibited by glucagon to a much greater relative extent than that observed during feeding. The results suggest that variations in the rate of lipogenesis over the diurnal cycle and during the first 24 hr of starvation could not be accounted for entirely by fluctuations in substrate availability. In contrast, changes which occurred subsequent to this (up to 43 hr of starvation) could be eliminated when lipogenic substrate was made more abundant. Longer periods of starvation were marked by a relative increase in the ability of glucagon to prevent the substrate-induced stimulation of lipogenesis.