Although initiation of translation at non-AUG codons occurs occasionally in prokaryotes and higher eukaryotes, it has not been reported in yeast until very recently. Evidence presented here shows that redundant ACG codons are recognized as alternative translation start sites for ALA1, the only gene in Saccharomyces cerevisiae coding for alanyl-tRNA synthetase. ALA1 is shown to be a bifunctional gene that provides both cytoplasmic and mitochondrial activities. Unlike most bifunctional genes that contain alternative in-frame AUG initiators, there is only one AUG codon, designated AUG1, close to the 5'-end of the ALA1 open reading frame. Transcriptional mapping identified three overlapping transcripts, with 5'-ends at positions 54, 105, and 117 nucleotides upstream of AUG1, respectively. Site-specific mutagenesis demonstrated that the cytoplasmic and mitochondrial functions of ALA1 are provided by two protein isoforms with distinct amino termini; that is, a short cytoplasmic form initiated at AUG1 and a longer mitochondrial isoform initiated at two upstream in-frame ACG codons, i.e. ACG(-25) and ACG(-24). These two ACG codons function redundantly in initiation of translation. Either codon can function in the absence of the other. The short transcript appears to serve as the template for the cytoplasmic form, whereas the longer transcripts are likely to code for both isoforms via alternative initiation. Because yeast ribosomes in general cannot efficiently recognize a non-AUG initiator, this unique feature of redundancy of non-AUG initiators in a single mRNA may in itself represent a novel paradigm for translation initiation from poor initiators.