Respiratory chain proteins encoded by the yeast mitochondrial genome are synthesized within the organelle. Mitochondrial mRNAs lack a 5' cap structure and contain long AU-rich 5' untranslated regions (UTRs) with many potential translational start sites and no apparent Shine-Dalgarno-like complementarity to the 15S mitochondrial rRNA. However, translation initiation requires specific interactions between the 5' UTRs of the mRNAs, mRNA-specific activators, and the ribosomes. In an initial step toward identifying potential binding sites for the mRNA-specific translational activators and the ribosomes, we have analyzed the effects of deletions in the 5' UTR of the mitochondrial COB gene on translation of COB transcripts in vivo. The deletions define two regions of the COB 5' UTR that are important for translation and indicate that sequence just 5' of the AUG is involved in selection of the correct start codon. Taken together, the data implicate specific regions of the 5' UTR of COB mRNA as possible targets for the mitochondrial translational machinery.