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The regulation of mitochondrial citrate metabolism has been investigated in oleaginous and non-oleaginous yeasts to ascertain its importance in controlling the rate of citrate efflux from mitochondria. The following observations were made :1. Citrate efflux from mitochondria of the oleaginous yeast Candida curuata D, in the presence of L-malate and pyruvate, was stimulated by adding ATP and reduced by AMP. In the non-oleaginous yeast, Candida utilis 359, there was very little stimulation of citrate efflux by ATP but it was reduced by AMP. These effects appeared to be generalized as similar results were obtained in an examination of eight further yeasts (seven oleaginous and one nonoleaginous).2. The effects of ATP and AMP were not observed in mitochondria whose metabolism had been inhibited by antimycin A and rotenone indicating the direct regulation of the citrate translocase was not involved.3. In C. curvata D, ATP increased the total mitochondrial citrate content and reduced that of 2-oxoglutarate whereas AMP had the reverse effect. In C. utilis 359, AMP had a similar effect but that of ATP was much smaller.4. To explain these observations the mitochondrial NADC-dependent isocitrate dehydrogenase was studied in a number of yeasts. The enzyme from oleaginous yeasts had a requirement for AMP for activity and was inhibited by ATP. In non-oleaginous yeasts the enzyme was active in the absence of AMP and increased in activity as the isocitrate concentration increased.5. The enzyme in C. curvata D was constantly more sensitive to increasing energy charge than that of the nonoleaginous yeast.These results indicate that the supply ofcitrate (and hence acetyl-CoA) to the cytosol is controlled by the activity of the intramitochondrial NAD + -dependent isocitrate dehydrogenase which in turn is regulated by adenine nucleotides. The sensitivity of this enzyme to the ATP/AMP ratio during lipogenesis is therefore an important control in the accumulation of lipid by yeasts.Work in our laboratory has been concerned with the biochemistry of various oleaginous yeasts and moulds which can accumulate large quantities of lipid [l, 21. When a nutrient, usually nitrogen, becomes depleted from the growth medium, protein and nucleic acid synthesis ceases but the excess carbon continues to be metabolised to lipid [3-51. Under such conditions, non-oleaginous yeasts still do not accumulate lipid. We have so far explained microbial oleaginicity [6] by showing that under conditions of nitrogen limitation, citrate accumulates in the mitochondria due primarily to a decrease in the intracellular AMP concentration which leads to a decline in the activity of the AMP-requiring, NAD +-dependent isocitrate dehydrogenase within the mitochondria. This citrate is then transported across the mitochondrial membrane in exchange for L-malate [7] and is cleaved in the cytosol by ATP:citrate lyase to yield acetyl-CoA (and oxaloacetate) from which fatty acids are synthesized. The presence of ATP :citrate lyase is confined to oleaginous yeasts and moulds K91...