The nucleotide sequence corresponding to codons for the 17-amino acid residues in the presumed targeting presequence for yeast mitochondrial malate dehydrogenase was removed by oligonucleotide-directed mutagenesis of the isolated gene (MDH1). Integrative transformation was used to insert the "leaderless" gene (mdhl-) into the MDH1 chromosomal locus of a strain containing a disrupted MDH1 gene. Expression of the mature form of malate dehydrogenase as a primary translation product was verified by demonstrating that the mature form is synthesized in mdhl- cells at the same rate as the precursor form in MDH1 cells in the presence of carbonyl cyanide m-chlorophenylhydrazone and by comparison of in vitro translation products of RNAs from mdhl- and MDH1 cells. Expression of mdhl- restores total cellular malate dehydrogenase activity to levels comparable to those in wild type cells and reverses the phenotype associated with strains containing MDH1 disruptions by restoring wild type rates of growth in media containing acetate as a carbon source. Immunochemical analyses and enzyme assays show comparable levels of malate dehydrogenase in the matrix fractions from mitochondria isolated from mdhl- and MDH1 cells and give no evidence for accumulation of the mature enzyme in the cytosol of mdhl- cells. These results indicate that the presequence for malate dehydrogenase is not essential for efficient mitochondrial localization or function in yeast.