1. 1. The effect of malate, succinate and malonate on citrulline synthesis in rat liver mitochondria was studied. 2. 2. When NH 4Cl was used as the source of ammonia, malate as well as succinate and malonate inhibited the citrulline formation by about 40–50% in mitochondria incubated with glutamate in State 4 as well as in uncoupled (+oligomycin) mitochondria with exogenous ATP as an energy source. The addition of either arginine to mitochondria incubated in State 4 or N-acetylglutamate to uncoupled mitochondria only slightly decreased the inhibition of citrulline formation by malate. 3. 3. The inhibitory effect of malate on citrulline formation was not observed in both toluene-treated and sonicated mitochondria as well as on the addition of n-butylmalonate into the suspension of mitochondria. 4. 4. When carbamoyl phosphate and ornithine were used as substrates for ornithine carbamoyltransferase the inhibitory effect of malate on citrulline production was observed. 5. 5. Malate decreased significantly the ornithine uptake by mitochondria incubated in the absence and presence of citrulline or N-acetylglutamate. 6. 6. The data presented indicate that with glutamate as substrate the inhibition of citrulline formation by malate may be not only due to a decline of N-acetylglutamate level as postulated by Meijer & Van Woerkom (1978) but also due to a decrease of mitochondrial ornithine uptake.