Class II tRNA synthetases have long been known to have quaternary structures of alpha, alpha2, alpha2beta2, and alpha4, depending on the amino acid specificity and the organism from which the synthetase was isolated. Even the quaternary structures of enzymes for the same amino acid show variations in evolution. The basis for these variations has not been understood. We report here that sequence manipulations of a structural motif (motif 1) characteristic of all class II tRNA synthetases can generate most of the evolutionary diversity of quaternary forms of class II synthetases. Thus, the principles elucidated here for quaternary structure assembly may be general.