Pneumolysin from Streptococcus pneumoniae was expressed in Escherichia coli as a glutathione S-transferase fusion protein and purified by affinity and hydroxylapatite chromatography. The purified recombinant pneumolysin (rPL), with a molecular mass of 53 kDa, had a specific activity of 3 x 10(5) hemolytic units per mg of protein on rabbit erythrocytes and reacted identically in immunodiffusion with the antisera against native pneumolysin. The rPL was used as a protein carrier to prepare conjugate vaccine with pneumococcal type 18C polysaccharide (PS18C). The PS18C was directly coupled to rPL by reductive animation or was indirectly coupled to rPL via a spacer molecule, adipic acid dihydrazide. The conjugates were nontoxic for mice and guinea pigs at 100 micrograms per dose. The immunogenicity and protective efficacy of both conjugates were tested in mice. A single dose of either of the vaccines elicited a rise in immunoglobulin G antibody production; after two booster injections of the vaccines, statistically significant booster responses (P < 0.001) to both rPL and PS18C were produced. The sera containing the antibodies to rPL were capable of neutralizing the hemolytic activity of rPL to rabbit erythrocytes and the cytotoxicity of rPL to bovine pulmonary endothelial cells. Immunization with the conjugate vaccines conferred statistically significant protection in mice against lethal challenge with type 18C pneumococci.