The tricarboxylic acid (TCA) cycle enzyme isocitrate dehydrogenase (IDH) and the glyoxylate bypass enzyme isocitrate lyase are involved in catabolism of isocitrate and play a key role in controlling the metabolic flux between the TCA cycle and the glyoxylate shunt. Two IDH genes icd1 and icd2 of Ralstonia eutropha HF39, encoding isocitrate dehydrogenase 1 (IDH1) and isocitrate dehydrogenase 2 (IDH2), were identified and characterized. Icd1 was functionally expressed in Escherichia coli, whereas icd2 was expressed in E. coli but no activity was obtained. Interposon-mutants of icd1 (HF39vicd1) and icd2 (HF39vicd2) of R. eutropha HF39 were constructed and their phenotypes were investigated. HF39vicd1 retained 43% of IDH activity, which was not induced by a cetate, and HF39vicd2 expressed 74% of acetate-induced IDH activity. Both HF39vicd1and HF39vicd2 kept the same growth rate on acetate as the wild-type. These data suggested that IDH1 is induced by acetate. The interposon-mutants HF39vicd1 and HF39vicd2 accumulated the same amount of poly(3-hydroxybutyric acid) as the wild-type.