Cytochrome P450 isolated from Bacillus subtilis (P450BSbeta; MW 48 kDa) catalyzes the hydroxylation of long-chain fatty acids at the alpha and beta positions using H(2)O(2) as an oxidant. Crystals of the substrate-free form of P450BSbeta belonging to the trigonal space group P3(2)21 or P3(1)21 were obtained by the sitting-drop vapour-diffusion method using a precipitate solution consisting of 10%(w/v) PEG 4000 and 50 mM MES pH 6.8. Another crystal form, belonging to the rhombohedral space group R3 or R32, was obtained from precipitate solution consisting of 10% PEG 4000, 0.15 mM magnesium acetate and 50 mM MES pH 6.5 in the presence of 2 mM myristic acid (substrate). Using synchrotron radiation, both P450BSbeta crystals diffracted to 2.5 A resolution. Bijvoet and dispersive anomalous difference Patterson maps show a clear peak corresponding to the haem iron.