Aminoacylation requires that an enzyme-bound aminoacyladenylate is brought proximal to the 3' end of a specific transfer RNA. In Escherichia coli alanyl-tRNA synthetase, the first 368 amino acids encode a domain for adenylate synthesis while sequences on the carboxyl-terminal side of this domain are required for much of the enzyme-tRNAAla binding energy. The 3' end of E. coli tRNAAla has been cross-linked to the enzyme, and sequence analysis showed that Lys-73 is the major site of coupling. A mutant enzyme with a Lys-73----Gln replacement has a 50-fold reduced kcat/Km (with respect to tRNAAla) for aminoacylation but has a relatively small alteration of its kinetic parameters for ATP and alanine in the adenylate synthesis reaction. The data provide evidence that the 3' end of tRNAAla binds to a site in the enzyme domain responsible for adenylate synthesis and that a residue (Lys-73) in this domain is important for a tRNAAla-dependent step that is subsequent to the synthesis of the aminoacyladenylate intermediate.