We have expressed the pneumolysin gene of Streptococcus pneumoniae in Bacillus subtilis, both from its own promoter and as a fusion protein. The level of expression of pneumolysin from its own promoter was low. The protein produced was hemolytically active. A higher level of expression (about 10 μg/ml of culture) was achieved when either one of two C-terminal fragments (corresponding to amino acids 265–471 and 55–471, respectively) or the entire coding part of the pneumolysin gene were fused to the promoter and signal sequence-coding region of the α-amylase gene of Bacillus amyloliquefaciens. The C-terminal fusion peptides reacted with antipneumolysin serum, but were not hemolytically active. In both cases most of the peptide remained cellassociated. When the entire pneumolysin gene was fused to the signal sequence, a hemolytically active form of pneumolysin could be detected, and most of the product was found in a processed form in the culture supernatant. The full-length pneumolysin secreted from B. subtilis was partially purified and used as antigen in an enzyme immunoassay with rabbit anti-pneumolysin serum.