The glyoxylate bypass genes aceA1 (isocitrate lyase 1, ICL1), aceA2 (isocitrate lyase 2, ICL2) and aceB1 (malate synthase, MS1) of Ralstonia eutropha HF39 were cloned, sequenced and functionally expressed in Escherichia coli. Interposon-mutants of all three genes (DeltaaceA1, DeltaaceA2 and DeltaaceB1) were constructed, and the phenotypes of the respective mutants were investigated. Whereas R. eutropha HF39DeltaaceA1 retained only 19% of ICL activity and failed to grow on acetate, R. eutropha HF39DeltaaceA2 retained 84% of acetate-inducible ICL activity, and growth on acetate was not retarded. These data suggested that ICL1 is in contrast to ICL2 induced by acetate and specific for the glyoxylate cycle. R. eutropha HF39DeltaaceB1 retained on acetate as well as on gluconate about 41-42% of MS activity and exhibited retarded growth on acetate, indicating the presence of a second hitherto not identified MS in R. eutropha HF39. Whereas in R. eutropha HF39DeltaaceA1 and R. eutropha HF39DeltaaceA2 the yields of poly(3-hydroxybutyric acid), using gluconate as carbon source, were significantly reduced, R. eutropha HF39DeltaaceB1 accumulated the same amount of this polyester from gluconate as well as from acetate as R. eutropha HF39.