4-Hydroxy-3-methyl-2-keto-pentanoate aldolase (asHPAL), an enzyme used in the synthesis of (2S,3R,4S)-4-hydroxyisoleucine, was crystallized in the absence and the presence of 2-ketobutyrate as one of its substrates by the sitting-drop vapour-diffusion method using PEG 400 as a precipitant. Crystals of asHPAL grown without and with 2-ketobutyrate diffracted to 1.60 and 1.55 Å resolution and belonged to space group C2, with unit-cell parameters a = 116.8, b = 88.2, c = 85.3 Å, β = 122.3° and a = 116.2, b = 88.1, c = 85.0 Å, β = 122.3°, respectively.