The gene encoding the histidine-tRNA synthetase (HTS1) has two in-frame translation start sites located 60 bp apart. One set of HTS1 transcripts (long) initiates upstream of both ATG codons, and the other set (short) initiates between the two ATG codons and therefore contains only the downstream ATG. A mutation that destroys the first AUG on the long message results in the Pet- (respiratory deficient) phenotype, but does not affect either the level of the cytoplasmic histidine-tRNA synthetase or viability. Mutations distal to the second ATG lead to loss of cytoplasmic synthetase function, lethality and respiratory deficiency. These phenotypes can be explained if the longer message were to encode the mitochondrial synthetase and the shorter message were to encode the cytoplasmic histidine-tRNA synthetase.