Valyl-tRNA synthetase (ValRS) strictly discriminates the cognate L-valine from the larger L-isoleucine and the isosteric L-threonine by the tRNA-dependent 'double sieve' mechanism. In this study, we determined the 2.9 Å crystal structure of a complex of Thermus thermophilus ValRS, tRNA(Val), and an analog of the Val-adenylate intermediate. The analog is bound in a pocket, where Pro41 allows accommodation of the Val and Thr moieties but precludes the lle moiety (the first sieve), on the aminoacylation domain. The editing domain, which hydrolyzes incorrectly synthesized Thr-tRNA(Val), is bound to the 3' adenosine of tRNA(Val). A contiguous pocket was found to accommodate the Thr moiety, but not the Val moiety (the second sieve). Furthermore, another Thr binding pocket for Thr-adenylate hydrolysis was suggested on the editing domain.