Aims: Detailed knowledge about the enzymes responsible for conversion of C-3 and C-4 compounds will be helpful to establish the bacterial strain Ralstonia eutropha as platform for the production of biotechnologically interesting compounds. Although various studies about these enzymes were accomplished in the past, some contradicting information about the enzyme pattern in this bacterium still exists. To resolve these discrepancies, the C-3/C-4 metabolism was reinvestigated after the genome sequence of this bacterium became available. Methods and Results: In silico analysis of genome sequence revealed putative genes coding for NAD(P)+-dependent malic enzymes (Mae), phoshoenolpyruvate carboxykinase (Pck), phosphoenolpyruvate carboxylase (Ppc), phosphoenolpyruvate synthase (Pps) and pyruvate carboxylase (Pyc). Reverse transcription PCR revealed constitutive expression of mae and pck genes, whereas no transcripts of pyc and ppc were found. Expression of active NADP+-dependent MaeB and Pck and absence of Pyc and Ppc was confirmed by spectrophotometric enzyme assays. Conclusions: The data reported in this study suggest that two enzymes, (i) MaeB and (ii) Pck, mediate between the C-3 and C-4 intermediates in R. eutropha H16. The enzymatic conversion of pyruvate into phosphoenolpyruvate (PEP) is catalysed by Pps, and an NADH+-dependent Mdh mediates the reversible conversion of malate and oxaloacetate. Significance and Impact of the Study: An increased knowledge of the enzymes mediating between C-3 and C-4 intermediates in R. eutropha will facilitate metabolic engineering.