Arabidopsis thaliana has three genes that encode distinct aconitases (ACO), but little is known about the function of each isoenzyme during plant development. In newly emerged seedlings of Arabidopsis, transcript and protein levels for ACO3 were selectively induced to yield more than 80% of total aconitase activity. Characterization of knock-out mutants for each of the three ACOs suggests a major role for only ACO3 in citrate metabolism. The aco3 mutant displayed delayed early seedling growth, altered assimilation of [14C]acetate feeding, and elevated citrate levels, which were nearly 4-fold greater than in wild type, aco1 or aco2. However, both ACO1 and ACO2 are active in seedlings as shown by inhibition of aco3 growth by the toxin monofluoroacetate, and altered [14C]acetate assimilation and metabolite levels in aco1 and aco2. Relative levels of fumarate and malate differed between aco2 and aco3 indicating metabolically isolated pools of these metabolites in seedlings. Our inability to enrich ACO protein through mitochondria isolation, and the reduced cytosolic ACO activity of the iron-sulfur center assembly mutant atm3-1, indicated a cytosolic localization of ACO3 in 3 day-old seedlings. Subsequently, we determined that more than 90% of ACO3 was cytosolic. We conclude that ACO3 is cytosolic in young seedlings and functions in citrate catabolism consistent with the operation of the classic glyoxylate and not direct catabolism of citrate within mitochondria.