Streptococcus pneumoniae, is a major human pathogen causing diseases ranging from mild mucosal infections such as sinusitis, otitis media to life-threatening invasive diseases such as sepsis, meningitis, and pneumonia. The main biological niche for pneumococcus is the nasopharyngeal mucosa. Entry of the colonizing pneumococci into sterile organs such as middle ear, lungs or blood stream results in the onset of pneumococcal disease. The increased pneumococcal carriage and heightened risk of invasive pneumococcal disease associated with cigarette smoke (CS) exposure are commonly attributed to CS-mediated alterations in the host immune function. Since the colonizing microflora including pneumococci are also exposed to bioactive chemicals in CS, we hypothesized that CS exposure induces expression of virulence determinants required for S. pneumoniae carriage and infection. This hypothesis is founded on our previous observations that CS enhances virulence of Staphylococcus aureus, a major respiratory pathogen that colonizes the nasopharyngeal mucosa.S. pneumoniae strain TIGR4 was exposed to different concentrations (0%, 1%, 5% and 10%) of cigarette smoke extract (CSE, 100%CSE was generated by bubbling smoke of 3 Marlboro cigarettes in 20ml Todd Hewitt broth) in 96-well polystyrene plate at 370C for 18h. After vigorous washing, biofilm-bound bacteria were stained with 1% crystal violet. We observed significantly increased biofilm formation in CSE-exposed TIGR4 in comparison to their medium-exposed counterparts. CS-mediated induction of pneumococcal biofilm formation may be correlated with the increased pneumococcal carriage rate in individuals exposed to CS.To delineate the molecular mechanisms underlying CS-mediated induction of pneumococcal virulence we exposed TIGR4 to 0 % or 25% CSE for 2h. The qRT-PCR-based comparison revealed that CS exposure upregulates expression of lytA (autolysin) and capsular polysaccharide (cps). Pneumococcal autolysin is a key virulence factor involved in biofilm formation as well as immune evasion while the antiphagocytic activity of capsular polysaccharide is important for pneumococcal survival in blood. We also observed CS-mediated down regulation of pneumolysin (plyA) involved in the host cell lysis by forming pores in membranes.Overall, our results imply that exposure to cigarette smoke induces specific pneumococcal virulence factors necessary for colonization. However, confirmation of these in vitro results in a mouse model of respiratory tract infection is warranted.