1. The objective of this study was to determine the separate contributions of exogenous substrate and of kidney tissue to the support of function and metabolism in the isolated perfused rat kidney. The effects of the addition of L(+) [U-14C]lactate or D[U-14C]glucose either to a specially prepared substrate-free albumin (SFA) or to Fr. V bovine serum albumin (Fr. V-BSA) were compared. The Fr. V-BSA has significant quantities of lactate, citrate and free fatty acids associated with it. 2. Perfusion of the rat kidney with the Krebs-Ringer bicarbonate solution containing SFA, without addition of exogenous substrate, resulted in a lower % Na+ reabsorption (approximately 43%) than when the perfusions contained Fr. V-BSA (approximately 80%). Thus, kidney tissue can support at most 45% of Na+ reabsorption, while the substrates associated with the Fr. V-BSA can support approximately 30% of Na+ reabsorption. When the initial concentration of L(+)lactate in the perfusate containing SFA was progressively raised from 0 to 10 mM, % Na+ reabsorption increased to between 85 and 90%. 3. The apparent Km (0-59 mM) and the Vmax (0-67 micronmole g-1. min-1) for lactate oxidation in the presence of SFA were both significantly lower than when Fr. V-BSA was present (Km = 2-0 mM; Vmax = 1-1 micronmole g-1. min-1). The lower Km is interpreted as being due to the removal of substances from the Fr. V-BSA which competitively inhibit either the uptake or oxidation of lactate; the lower Vmax is considered to be related to the lower rate of Na+ reabsorption when SFA is present. 4. Addition of glucose enhanced gomerular filtration rate in the presence of both types of albumin. The resulting increase in the filtered load of Na+ in the presence of glucose was associated with either no change (Fr. V-BSA) or an increase (SFA) in fractional Na+ reabsorption. Although absolute Na+ reabsorptive rate was greater in the presence of glucose than in the presence of lactate, the oxidation rate of glucose, on a carbon-atom basis, was less than 50% of the oxidation rate of lactate. 5. The metabolism of glucose may regulate the permeability characteristics of the glomerulus and the tubular epithelium: by contrast, the high oxidation rate of lactate suggests it can provide direct support for a major fraction of the Na+ actively absorbed.