The gene of V-1-ATPase B subunit from the thermophilic eubacterium Thermus thermophilus has been cloned and the protein overproduced in Escherichia coli. The purified protein, with a molecular weight of 53.2 kDa, was crystallized from 10% (w/v) polyethylene glycol 1000, 120 mM magnesium chloride, and 100 mM Na-tricine, pH 8.0, by the vapor diffusion method. The crystals diffracted X-rays beyond 3.5 Angstrom on a synchrotron radiation source. The crystals belong to the monoclinic space group C2, with unit cell dimensions of a = 153.1 Angstrom, b = 129.6 Angstrom, c = 92.7 Angstrom, and beta = 100.3 degrees. Assuming that three or four molecules are contained in an asymmetric unit, the V-M value is calculated as 2.8 or 2.1 Angstrom (3)/Da, respectively.