The sub-cellular localisation of enzymes has been defined by latency analysis, and fractionation by differential centrifugation, in cell-free extracts prepared from the mycelium of Aspergillus nidulans by growth in the presence of 2-deoxyglucose followed by treatment with a mixture of beta-glucuronidase, sulphatase and beta-glucanase and exposure to N2 cavitation at 5.2 PMa. In such extracts pyruvate carboxylase and NAD-dependent and NADP-dependent glutamate dehydrogenases are exclusively localised in the cytosol whereas all the other enzymes studied have sub-cellular localisation patterns similar to those described for mammalian liver. Electrophoretic analysis has established the presence of unique mitochondrial and cytosolic isoenzymes for many of the enzymes, e.g. NAD--malate dehydrogenase, NADP--isocitrate dehydrogenase, glutamate/oxaloacetate transaminase, fumarase, which show a marked extent of incomplete latency and the presence of significant activity in the mitochondrial and cytosolic fractions prepared by differential centrifugation. A novel method is described for detection of citrate synthase activity following electrophoresis of the cell-free extract. Application of this method confirms the absence of a unique cytosolic isoenzyme of citrate synthase and hence shows that citrate synthase activity detected in the soluble fraction results from damage to the mitochondria during isolation. A scheme is proposed on the basis of these data to describe the organisation of lipid and amino acid synthesis from glucose in an organism which possesses a cytosolic pyruvate carboxylase.