We have characterized a mutation that affects the tRNAAsp coded by yeast mitochondrial DNA. Comparison of the DNA sequences of the tRNAAsp gene from a wild type strain and the mutant demonstrates that the mutant differs by a C to U base change in position 72 of the tRNA. This mutation abolishes mitochondrial protein synthesis, presumably because the tRNAAsp made from this gene cannot be charged with aspartic acid (FAye, G., Bolotin-Fukuhara, M., and Fukuhara, H. (1976) in The Genetics and Biogenesis of Chloroplasts and Mitochondria (Bucher, C. T., Neupert, W., Sebalt, W., and Werner, S., eds) pp. 547-555, North Holland Publishing Co., Amsterdam). It also reduces the amount of tRNAAsp transcripts in the mutant as compared to the wild type.