Saccharomyces cerevisiae accumulates L-malic acid but only minute amounts of fumaric acid. A 13C-nuclear magnetic resonance study following the label from glucose to L-malic acid indicates that the L-malic acid is synthesized from pyruvic acid via oxaloacetic acid. previously published studies, a cytosolic reductive pathway leading from pyruvic acid via oxaloacetic acid to L-malic acid is responsible for the L-malic acid production in yeast. The non-production of fumaric acid can be explained by the conclusion that, in the cell, cytosolic fumarase catalyzes the conversion of fumaric acid to L-malic acid but not the reverse. This conclusion is based on the following findings. (a) The cytosolic enzyme exhibits a 17-fold higher affinity towards fumaric acid than towards L-malic acid; the K(m) for L-malic acid is very high indicating that L-malic acid is not an in vivo substrate of the enzyme. (b) Overexpression of cytosolic fumarase does not cause accumulation of fumaric acid (but rather more L-malic acid). (c) According to 13C NMR studies there is no interconversion of cytosolic L-malic and fumaric acids.