The subcellular localization of the four characteristic enzymes of the methylcitric acid cycle was studied with glucose-grown as well as n-alkane-grown cells of Yarrowia lipolytica. Microsomes and peroxisomes showed no cycle enzyme activities. The four cycle enzymes were constitutively localized in mitochondria, with the exception of the dual localization of the fourth enzyme, 2-methylisocitrate lyase, in mitochondria and cytoplasm, where the lyase may function to supply pyruvate (the end-product of the catabolism of the propionate residue) to various reactions.