Previous studies have shown that in Saccharomyces cerevisiae the mitochondrial and cytoplasmic forms of alanyl-tRNA synthetase are encoded by a single nuclear gene, ALA1, through alternative use of in-frame successive ACG triplets and a downstream AUG triplet. Here we show that despite the obvious participation of the non-AUG-initiated leader peptide in mitochondrial localization, the leader peptide per se cannot target a cytoplasmic passenger protein into mitochondria under normal conditions. Functional mapping further shows that an efficient targeting signal is composed of the leader peptide and an 18-residue sequence downstream of Met1. Consistent to this observation, overexpression of the cytoplasmic form enables it to overcome the compartmental barrier and function in the mitochondria as well, but deletion of as few as eight amino acid residues from its amino-terminus eliminates such a potential. Thus, the sequence upstream of the first in-frame AUG initiator not only carries an unusual initiation site, but also contributes to a novel pattern of protein expression and localization.