Nicotine has a deleterious impact on gingival fibroblast cell viability, adhesion, and migration. Less is known regarding the effect of cotinine, the main metabolite of nicotine, on such processes. The objective of this study was to determine if cotinine affects the adhesion or migration of Human Gingival Fibroblasts (HGF) in culture. HGF were treated with nicotine or cotinine at several concentrations and dose-response cytotoxicity was determined by MTT assay. The effects of nicotine and cotinine on HGF adhesion were measured colorimetrically and cell migration was determined using the scratch wound assay. The number of HGF oriented parallel to the wound edge at 24 hours was counted using phase contrast images. Data were analyzed using ANOVA and Dunnet's multiple comparison post-test. At the highest concentrations of cotinine (640 ng/ml) and nicotine (400 µg/ml) both HGF survival and cell adhesion were significantly inhibited (p<0.01). By scratch wound assay HGF migration from the wound edge at 24 hours was significantly inhibited by 320 ng/ml (p<0.001) and 640 ng/ml (p<0.001) cotinine, and by 400 µg/ml (p<0.01) nicotine. HGF migration was significantly inhibited by 80 ng/ml (p<0.05), 320 ng/ml (p<0.01) and 640 ng/ml (p<0.001) cotinine and by 100 µg/ml (p<0.01), 200 µg/ml (p<0.01) and 400 µg/ml (p<0.001) nicotine at 48 hours. Significantly more HGF were oriented parallel to wound edge with pre treatment of 10 ng/ml cotinine or 50 µg/ml nicotine before wounding (p<0.001). In HGF exposed to nicotine (400 µg/ml) or cotinine (640 ng/ml), cell survival, cell adhesion, and migration were significantly decreased, but cell polarity was not affected. These concentrations are within ranges of serum levels in smokers, providing evidence that multiple cellular aspects of wound healing are compromised in tobacco users.