The membrane-bound GPI-anchored zinc metalloproteinase leishmanolysin purified from Leishmania major promastigotes has been crystallized in its mature form. Two crystal forms of leishmanolysin have been grown by the vapor diffusion method using 2-methyl-2,4-pentanediol as the precipitant. Macroseeding techniques were employed to produce large single crystals. Protein microheterogeneity in molecular size and charge was incorporated into both crystal forms. The tetragonal crystal form belongs to the space group P4(1)2(1)2 or the enantiomorph P4(3)2(1)2, has unit cell parameters of a = b = 63.6 A, c = 251.4 A, and contains one molecule per asymmetric unit. The second crystal form is monoclinic, space group C2, with unit cell dimensions a = 107.2 A, b = 90.6 A, c = 70.6 A, beta = 110.6 degrees, and also contains one molecule per asymmetric unit. Both crystal forms diffract X-rays beyond 2.6 A resolution and are suitable for X-ray analysis. Native diffraction data sets have been collected and the structure determination of leishmanolysin using a combination of the isomorphous replacement and the molecular replacement methods is in progress.