We report here that tyrosyl-tRNA synthetase from the eukaryotic pathogen Pneumocystis carinii is a 370 amino acid polypeptide with characteristic elements of a class I aminoacyl-tRNA synthetase and aligns with the prokaryotic tyrosyl-tRNA synthetases in the class-defining active site region, including the tRNA acceptor helix-binding region. The expressed enzyme is a dimer that aminoacylates yeast tRNA but not Escherichia coli tRNA(Tyr). Like most tRNAs, prokaryotic tyrosine tRNAs have a G1.C72 base pair at the ends of their respective acceptor helices. However, the eukaryote cytoplasmic tyrosine tRNAs have an uncommon C1.G72 base pair. We show that P. carinii tyrosyl-tRNA synthetase charges a seven base pair hairpin microhelix (microhelixTyr) whose sequence is derived from the acceptor stem of yeast cytoplasmic tRNATyr. In contrast, the enzyme does not charge E. coli microhelixTyr. Changing the C1.G72 of yeast microhelixTyr to G1.C72 abolishes charging by the P. carinii tyrosyl-tRNA synthetase. Conversely, we found that E. coli tyrosyl-tRNA synthetase can charge an E. coli microhelixTyr and that charging is sensitive to having a G1.C72 rather than a C1.G72 base pair. The results demonstrate that the common structural framework of homologous tRNA synthetases has the capacity to coadapt to a transversion in a critical acceptor helix base pair and that this coadaptation can account for species-selective microhelix aminoacylation. We propose that species-selective acceptor helix recognition can be used as a conceptual basis for species-specific inhibitors of tRNA synthetases.