The class-defining active site domain of the 10 class II tRNA synthetases is well conserved and, based on the crystal structure of aspartyl-tRNA synthetase, approaches the end of the tRNA acceptor stem from the major groove side of the helix. Paradoxically, for the class II alanyl-tRNA synthetase (AlaRS), aminoacylation is dependent on minor groove recognition of an acceptor helix G3.U70 base pair. Additional contributions to aminoacylation efficiency are made by the A73 "discriminator" base and G2.C71 base pair located at the end of the acceptor stem. Using microhelix substrates containing only the first four base pairs of the alanine tRNA acceptor helix, we demonstrated that the catalytic center of AlaRS with the three class-defining sequence motifs contains determinants for recognition of A73 and G2.C71. However, this structural unit does not discriminate between different base pairs at the critical 3.70 position. Discrimination at G3.U70 was mapped to a 76 amino acid polypeptide outside the catalytic center. We propose that the G3.U70 recognition motif is a structural appendage that folds back to the catalytic center to make contact with the bound acceptor stem. A "fold-back" appendage provides a specific mechanism for minor groove recognition of the acceptor helix by a class II tRNA synthetase.