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Objective The present study assessed the effect of smoking on clinical, microbiological and immunological parameters in an experimental gingivitis model. Material and Methods Twenty-four healthy dental students were divided into two groups: smokers ( n = 10); and nonsmokers ( n = 14). Stents were used to prevent biofilm removal during brushing. Visible plaque index ( VPI) and gingival bleeding index ( GBI) were determined 5- on day -7 (running phase), baseline, 21 d (experimental gingivitis) and 28 d (resolution phase). Supragingival biofilm and gingival crevicular fluid were collected and assayed by checkerboard DNA- DNA hybridization and a multiplex analysis, respectively. Intragroup comparison was performed by Friedman and Dunn's multiple comparison tests, whereas the Mann-Whitney U-test was applied for intergroup analyses. Results Cessation of oral hygiene resulted in a significant increase in VPI, GBI and gingival crevicular fluid volume in both groups, which returned to baseline levels 7 d after oral hygiene was resumed. Smokers presented lower GBI than did nonsmokers ( p < 0.05) at day 21. Smokers had higher total bacterial counts and higher proportions of red- and orange complex bacteria, as well as lower proportions of Actinomyces spp., and of purple- and yellow-complex bacteria ( p < 0.05). Furthermore, the levels of key immune-regulatory cytokines, including interleukin ( IL)-8, IL-17 and interferon-γ, were higher in smokers than in nonsmokers ( p < 0.05). Conclusion Smokers and nonsmokers developed gingival inflammation after supragingival biofilm accumulation, but smokers had less bleeding, higher proportions of periodontal pathogens and distinct host-response patterns during the course of experimental gingivitis. [ABSTRACT FROM AUTHOR]