Mycobacteria synthesize mycothiol (MSH) as a low-molecular-weight thiol that protects against oxidative stress in a similar role to that of glutathione in many other species. The absence of MSH in mammals suggests that enzymes from its biosynthetic pathway in Mycobacterium tuberculosis could be useful targets for drug design. The gene for MshB (Rv1170), the enzyme that catalyses the second step in MSH biosynthesis in M. tuberculosis, has been cloned and the protein has been expressed in Escherichia coli both in native and SeMet-substituted forms and crystallized in two crystal forms. One of these, prepared in the presence of beta-octylglucoside as a key additive, is suitable for high-resolution X-ray structural analysis. The crystals are orthorhombic, with unit-cell parameters a = 71.69, b = 83.74, c = 95.65 A, space group P2(1)2(1)2(1) and two molecules in the asymmetric unit. X-ray diffraction data to 1.9 A resolution have been collected.