The addition of D-glucose to renal proximal tubule cells reduces phosphate transport and in conditions of limited phosphate availability reduces mitochondrial respiration, oxidative phosphorylation and ATP content, a phenomenon called the Crabtree effect. The present study examined the effects of D-glucose on cellular content of inorganic phosphate, phosphomonoesters, and ATP. These studies were performed in suspensions of canine proximal tubules using 31P NMR spectroscopy. When the extracellular medium contained 0.4 mM phosphate, addition of 8.3 mM D-glucose to the perifusion fluid significantly lowered cell content of inorganic phosphate (by 31%), phosphomonoesters (by 23%) and ATP (by 25%). Addition of a similar concentration of mannitol had no significant effect on these measured parameters. Removal of inorganic phosphate from the extracellular medium in the absence of glucose reduced the cellular content of inorganic phosphate by only 9%. These data indicate that a reduction in cellular phosphate occurs during the Crabtree effect. The glucose effect on cellular phosphates appears to be an inhibition of phosphate uptake and an enhancement of phosphate efflux. The data do not support the hypothesis that glucose induces an increase in sugar monophosphates.