Although the well-recognised predisposition of cigarette smokers to the development of severe pneumococcal disease may be attributable to impairment of local host defences, less is known about the direct effects of smoke exposure on airway pathogens, or their virulence factors. In the current study, we have investigated the effects of cigarette smoke condensate (CSC) on biofilm formation by Streptococcus pneumoniae, and on the pore-forming activity of its major toxin, pneumolysin. Biofilm formation following exposure of the pneumococcus to CSC (20-160 μg·mL(-1)) was measured using a crystal violet-based spectrophotometric procedure, while the pore-forming activity of recombinant pneumolysin was determined by a fura-2/acetoxymethyl ester-based spectrofluorimetric procedure to monitor the uptake of extracellular Ca(2+) by isolated human neutrophils. Exposure of the pneumococcus or pneumolysin to CSC resulted in significant dose-related augmentation of biofilm formation (p≤0.05 at 80 and 160 μg·mL(-1)) and substantial attenuation of the pore-forming interactions of pneumolysin, respectively. Augmentation of biofilm formation and inactivation of pneumolysin as a consequence of smoking are likely to favour microbial colonisation and persistence, both being essential precursors of pneumococcal disease.