The effects of various short-chain fatty acids, carboxylic acids, and amino acids on NADH fluorescence and oxygen consumption (QO2) of rabbit proximal tubule suspensions were determined. The short-chain fatty acids were the most effective substrates in increasing NADH fluorescence and QO2, followed by the carboxylic acids and amino acids. All of the substrates tested that increased NADH fluorescence proportionally increased QO2. This implies that the primary effect of these substrates was to increase QO2 by increasing the delivery of reducing equivalents to NAD and not by stimulating ATP hydrolysis directly. The relative affinity of several substrates to increase NADH fluorescence was also determined. The short-chain fatty acids had the highest affinity (10 microM range) followed by the carboxylic acids (100 microM range). These data demonstrate that the metabolic rate and NADH redox state of the renal cortical cell is very sensitive to the type of metabolic substrate available.