Previous studies have shown that translation of mrna for yeast glycyl-tRNA synthetase is alternatively initiated from UUG and a downstream AUG initiation codon. Evidence presented here shows that unlike an AUG initiation codon, efficiency of this non-AUG initiation codon is significantly affected by its sequence context, in particular the nucleotides at positions -3 to -1 relative to the initiation codon. A/A/R (R represents A Or G) and C/G/C appear to be the most and least favorable sequences at these positions, respectively. Mutation of the native context sequence -3 to -1 from AAA to CGC reduced translation initiation from the UUG codon up to 32-fold and resulted in loss of mitochondrial respiration. although an AUG initiation codon is, in general, unresponsive to context changes in yeast, an AAA (-3 to -1) to CGC mutation still reduced its initiating activity up to 8-fold under similar conditions. these results suggest that sequence context is more important for translation initiation in yeast than previously appreciated.