The class II Escherichia coli and human alanyl-tRNA synthetases cross-acylate their respective tRNAs and require, for aminoacylation, an acceptor helix G3:U70 base pair that is conserved in evolution. We report here the primary structure and expression in the yeast Pichia of an active human alanyl-tRNA synthetase. The N-terminal 498 amino acids of the 968-residue polypeptide have substantial (41%) identity with the E. coli protein. A closely related region encompasses the class-defining domain of the E. coli enzyme and includes the part needed for recognition of the acceptor helix. As a result, previously reported mutagenesis, modeling, domain organization, and biochemical characterization on the E. coli protein appear valid as a template for the human protein. In particular, we show that both the E. coli enzyme and the human enzyme purified from Pichia aminoacylate 9-base pair RNA duplexes whose sequences are based on the acceptor stems of either E. coli or human alanine tRNAs. In contrast, the sequences of the two enzymes completely diverge in an internal portion of the C-terminal half that is essential for tetramer formation by the E. coli enzyme, but that is dispensable for microhelix aminoacylation. This divergence correlates with the expressed human enzyme behaving as a monomer. Thus, the region of close sequence similarity may be a consequence of strong selective pressure to conserve the acceptor helix G3:U70 base pair as an RNA signal for alanine.