The 4-hydroxyphenylacetate (4HPA) 3-monooxygenase enzyme catalyzes the hydroxylation of 4HPA to 3,4-dihydroxyphenylacetate in the initial step of the degradation pathway of 4HPA. This enzyme consists of two components: an oxygenase (HpaB) and a reductase (HpaC). HpaB hydroxylates 4HPA using an oxygen molecule and a reduced flavin, which is supplied by HpaC. HpaB from Thermus thermophilus HB8 was overexpressed in Escherichia coli and crystallized. Crystals of HpaB were grown in 0.4 M 1,6-hexanediol, 0.1 M sodium acetate pH 5.0 and 25% (v/v) glycerol and diffracted X-rays to a resolution of 1.60 A. The crystals belong to the orthorhombic space group I222, with unit-cell parameters a = 91.8, b = 99.6, c = 131.1 A. The asymmetric unit volume provides space for only one subunit of the tetrameric HpaB molecule, giving a Matthews coefficient V(M) of 2.8 A3 Da(-1) and a solvent content of 55.1%. Platinum-derivatized crystals of HpaB were prepared by soaking native crystals in a solution containing 1 mM ammonium tetrachloroplatinate(II) for 1 d and diffracted X-rays to a resolution of 2.50 A. MAD data were successfully collected for structural determination using these crystals.