We have purified the product of the NAM2 gene, the mitochondrial leucyl-tRNA synthetase, from yeast mitochondria. The purified protein cross-reacts with antibodies raised against the product of a LacZ/NAM2 gene fusion and antibodies raised against the purified Escherichia coli leucyl-tRNA synthetase. The mass as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is about 100 kDa, consistent with the size predicted by the gene sequence (102 kDa). The N-terminal sequence of the protein has been determined and shows that the first nine amino acids predicted by the gene sequence have been removed, probably during transport into the mitochondria.