A critical step in the faithful translation of genetic information is specific tRNA recognition by aminoacyl-tRNA synthetases. These enzymes catalyze the covalent attachment of particular amino acids to the terminal adenosine of cognate tRNA substrates. In general, there is one synthetase for each of the twenty amino acids and each enzyme must discriminate against all of the cellular tRNAs that are specific for the nineteen noncognate amino acids. Primary sequence information combined with structural data have resulted in the division of the twenty synthetases into two classes. In recent years, several high-resolution co-crystal structures along with biochemical data have led to an increased understanding of tRNA recognition by synthetases of both classes. The anticodon sequence and the amino acid acceptor stem are the most common locations for critical recognition elements. This review will focus on acceptor stem discrimination by class II synthetases. In particular, the results of in vitro aminoacylation assays and site-directed and atomic group mutagenesis studies will be discussed. These studies have revealed that even subtle atomic determinants can provide signals for specific tRNA aminoacylation.