Pneumolysin is a virulence factor from Streptococcus pneumoniae, a Gram-positive bacterial pathogen which causes human infections with a severe impact on mortality and morbidity worldwide. The enzyme belongs to a group of cholesterol-dependent cytolysins and interacts with its cholesterol receptor on target cells, leading to pneumolysin insertion into target-cell membranes and subsequently to pore formation and cell lysis. Pneumolysin has been overexpressed, purified and crystallized for X-ray diffraction studies. Crystals have been obtained in the presence of cholesterol in an effort to produce a three-dimensional structure of pneumolysin in its fully functional form with the enzyme bound to its activator. This is the first report of the crystallization of a cholesterol-dependent cytolysin in the presence of bound cholesterol. The vapor-diffusion method using ammonium sulfate as a precipitation agent was used to grow crystals in the presence of n-octyl-beta-D-glucopyranoside and phosphatidylcholine. Crystals of this 53 kDa molecule complexed with cholesterol diffracted X-rays to 3.3 A. The crystal unit cell has parameters a = b = 191.45, c = 66.16 A, alpha = beta = 90.0, gamma = 120 degrees and belongs to the trigonal space group P3. The determination of the three-dimensional structure of this pneumococcal cytolysin is in progress.