Glycine catabolism was studied in isolated rat liver mitochondria by measuring the release of 14CO2 from [1-14C]glycine. Mitochondria isolated from rats fed on a high-protein (60% casein) diet for 5 days showed an enhanced ability to catabolize glycine compared with mitochondria from rats fed on a normal-protein (15% casein) diet. Glycine catabolism was also stimulated in normal protein-fed rats if they ingested a single high-protein meal for 2 h before being killed, thus illustrating the rapid response of the glycine-cleavage system to protein intake. The stimulation of glycine catabolism in rats given a high-protein diet or meal was not evident if the mitochondria were incubated in the absence of P(i) (omitting ADP had no effect on the rate). Mitochondria from high-protein- and normal-protein-fed rats did not differ in their ability to accumulate glycine, a process which occurred far too rapidly to impose a limit on the rate of flux through the glycine-cleavage system. The stimulation of glycine catabolism by high-protein feeding was not associated with a change in mitochondrial matrix volume. Furthermore, mitochondria from rats fed on a high-protein meal maintained an enhanced ability to catabolize glycine compared with those from rats fed on a normal-protein meal when incubated in hypo-osmotic solutions of very low osmolarity. When mitochondria from high-protein- or normal-protein-fed rats were maximally activated by incubation in the presence of 0.25 microM-Ca2+, the rates of glycine catabolism were high, but similar, showing that the stimulation of glycine catabolism by high-protein feeding does not involve an increase in the total capacity of the system. These findings show that hepatic glycine catabolism is stimulated rapidly by high-protein feeding, a response that we suggest is involved in the disposal of the excess glycine in the diet.