In order to obtain information (1) on the quantitative contribution of various circulating substrates to renal metabolism and (2) on the relative importance of net luminal and basolateral transport for substrate uptake, we have precisely quantified the renal blood flow, the urinary flow, and the rates of substrate handling by the kidney of anesthetized fed and 72-hour-starved rats. For this, the concentration of twelve metabolites were simultaneously measured in arterial and venous whole blood and plasma as well as in urine of each rat thanks to the use of microassays based on enzymatic cycling. In fed rats, the main potential energy sources were glucose and lactate followed by fatty acids, ketone bodies, citrate and glycerol. Starvation caused a large increase in renal uptake and metabolism of fatty acids, ketone bodies, glutamine and glycerol, and a large inhibition of lactate utilization. The net peritubular uptake of acetoacetate, citrate, glycerol and free fatty acids demonstrated in both nutritional states was increased by starvation only for glycerol and free fatty acids; net peritubular efflux of both beta-hydroxybutyrate and ammonium ions was stimulated whereas that of glutamine was converted into net peritubular uptake by starvation.