Malate dehydrogenase from the syntrophic propionate-oxidizing bacterium strain MPOB was purified 42-fold. The native enzyme had an apparent molecular mass of 68 kDa and consisted of two subunits of 35 kDa. The enzyme exhibited maximum activity with oxaloacetate at pH 8.5 and 60 degrees C. The Ka for oxaloacetate was 50 microM and for NADH 30 microM. The Km values for L-malate and NAD were 4 and 1.1 mM, respectively. Substrate inhibition was found at oxaloacetate concentrations higher than 250 microM. The N-terminal amino acid sequence of the enzyme was similar to the sequences of a variety of other malate dehydrogenases from plants, animals and micro-organisms.