Rat lung mitochondrial preparations were incubated in the preasence of pyruvate and malate. The principal metabolic products measured were citrate and CO2. Citrate formation from pyruvate was found to be dependent on the presence of malate. Significant citrate was formed in the presence of isocitrate and the rate of citrate formation was increased by the addition of pyruvate. Small amounts of citrate were formed by lung mitochondrial preparations in the presence of 2-oxoglutarate and succinate only after the addition of pyruvate. The level of acetyl-CoA was significantly greater in the presence of pyruvate than in the presence of pyruvate plus malate. The addition of malate to lung mitoochondrial preparations increased 14CO2 production from [2-14C] pyruvate into malate and citrate. A low level of pyruvate-dependent H14CO3-incorporation into acid-stable products was observed, principally citrate and malate, but this rate did not exceed 5% of the rate of net citrate formation in the presence of malate and pyruvate. The capacity of rate lung mitochondria to form oxaloacetate from pyruvate alone in vitro is very limited, and would appear to cast doubt on a major role of pyruvate carboxylase in citrate formation. It is concluded that the rate of citrate formation from pyruvate is limited by the availability of intramitochondrial oxaloacetate and the rate of citrate efflux across the mitochondrial membrane.