Using benzoic acid as substrate, this study tested the hypothesis that capacity limitation of glycine conjugation in vivo is due to substrate-induced depletion of hepatic cosubstrates (i.e., ATP, coenzyme A, and glycine) utilized in the conjugation reaction. Benzolyglycine formation was investigated by following the disappearance of benzoic acid from blood and appearance of benzoylglycine in blood and urine after administration of sodium benzoate (0.2-2 mmol/kg, iv) to anesthetized rats whose urine formation was stimulated by mannitol administration. Capacity limitation of glycine conjugation is indicated by (a) the gradual dose-dependent reduction of benzoate blood clearance from 39 ml/min/kg at 0.2 mmol/kg benzoate to 3.7 ml/min/kg at 2 mmol/kg, and (b) the tendency to attain maximal blood levels and urinary excretion rates of benzoylglycine after administration of 0.5-1 mmol/kg benzoate. The maximal urinary excretion rate of benzoylglycine after benzoylglycine administration exceeded the maximal excretion rate of endogenously formed benzoylglycine (approximately 5 mumol/kg/min) 5-fold. This suggests that the urinary excretion rate of endogenously formed benzoylglycine reflects the rate of its formation. Benzoate depleted hepatic glycine (to 40%) and coenzyme A (to 14%) in a dose-dependent fashion; however, it did not change ATP levels in liver. The pattern of this dose-dependent cosubstrate depletion suggests that benzoate primarily causes consumption of hepatic glycine which, at high substrate dosage, leads to marked depletion of coenzyme A in the liver. Thus, these observations indicate that capacity-limited glycine conjugation may be due to limited availability of glycine and coenzyme A for the conjugation process.