Saccharomyces cerevisiae accumulates L-malic acid through a cytosolic pathway starting from pyruvic acid and involving the enzymes pyruvate carboxylase and malate dehydrogenase. In the present study, the role of malate dehydrogenase in the cytosolic pathway was studied. Overexpression of cytosolic malate dehydrogenase (MDH2) under either the strong inducible GALLO or the constitutive PGK promoter causes a 6- to 16-fold increase in cytosolic MDH activity in growth and production media and up to 3.7-fold increase in L- malic acid accumulation in the production medium. The high apparent K(m) of MDH2 for L-malic acid (11.8 mM) indicates a low affinity of the enzyme for this acid, which is consistent with the cytosolic function of the enzyme and differs from the previously published K(m) of the mitochondrial enzyme (MDH1, 0.28 mM). Under conditions of MDH2 overexpression, pyruvate carboxylase appears to be a limiting factor, thus providing a system for further metabolic engineering of L-malic acid production. The overexpression of MDH2 activity also causes an elevation in the accumulation of fumaric acid and citric acid. Accumulation of fumaric acid is presumably caused by high intracellular L-malic acid concentrations and the activity of the cytosolic fumarase. The accumulation of citric acid may suggest the intriguing possibility that cytosolic L-malic acid is a direct precursor of citric acid in yeast.