Previous investigators found that cyanide (CN-) is a potent inhibitor of renal Na+ transport, while the uncoupling agent 2,4-dinitrophenol (DNP) and fructose (both which lower ATP levels) are weak transport inhibitors. To examine the disparate effects of these substances measurements of ATP were performed, using 31P NMR, while simultaneously monitoring renal Na+ transport. Infusion of CN-, DNP, and fructose lowered whole kidney ATP levels by about the same extent (30%) while only CN- inhibited Na+ transport. This may be due to the fact that CN- has a potent action on the thick ascending limb of Henle, while fructose and DNP may have a more proximal action. Alternatively, ATP turnover may be a more important determinant of transport than ATP concentrations. Saturation transfer experiments were performed to measure Pi-ATP flux. Unilateral nephrectomy, high protein feeding, and methylprednisolone were used to stimulate metabolism and transport. The rate of Pi-ATP flux was 20.1 mumol/min/g. However, because oxygen consumption was stimulated, the ATP/O ratio was 0.85, considerably less than the theoretical value of 3. Finally, atrial natriuretic factor, which increased Na+ transport, had no effect on Pi-ATP flux. The results raise the possibility that the saturation transfer technique does not detect all Pi-ATP flux, especially when renal metabolism is stimulated.