It was previously shown that ALA1, the only alanyl-tRNA synthetase (AlaRS) gene in Saccharomyces cerevisiae, codes for two functionally exclusive protein isoforms through alternative initiation at two consecutive ACG codons and an in-frame downstream AUG. We reported here the cloning and characterization of a homologous gene from Candida albicans. Functional assays show that this gene can substitute for both the cytoplasmic and mitochondrial functions of ALA1 in S. cerevisiae, and codes for two distinct protein isoforms through alternative initiation from two in-frame AUG triplets 8-codons apart. Unexpectedly, while the short form acts exclusively in cytoplasm, the longer form provides function in both compartments. Similar observations are made in fractionation assays. Thus, the AlaRS gene of C. albicans has evolved an unusual pattern of translation initiation and protein partitioning, and codes for protein isoforms that can aminoacylate isoaccepting tRNAs from a different species and from across cellular compartments.