Abstract Cell-free extracts prepared from mycelium of Penicillium chrysogenum Q176 oxidized citrate, cis-aconitate, and isocitrate. In addition, citrate was formed from oxalacetic acid and a 2-carbon fragment in the presence of fluoroacetate. When citrate was metabolized aerobically by these extracts, one-half mole of oxygen uptake was observed for every mole of α-ketoglutarate formed. Carbon dioxide evolution was equivalent to the citrate dissimilated and, in crude extracts, far in excess of the keto acids formed. Under anaerobic conditions, citrate could be dissimilated quantitatively to glutamate and carbon dioxide by a TPN-mediated pathway, and the amount of citrate dissimilated was dependent on the ammonium-ion concentration. This pathway explains the “excess” carbon dioxide formed when citrate is dissimilated aerobically. The presence of isocitritase activity in these extracts was confirmed. This enzyme has a pH optimum of 6.0–6.2 and a bivalent metal-ion requirement satisfied by either magnesium or manganese.