The aim of the present study was to examine: (1) the effects of nicotine on human gingival fibroblasts (HGF); (2) differences between smokers (> or = 10 cigarettes/day at least for 5 years) and non-smokers; (3) differences between patients of different age. HGF were obtained, through biopsies during periodontal surgical procedures, from 15 patients which were divided in 4 groups: 4 patients, smokers aged < or = 25 years; 4 patients, non-smokers aged < or = 25 years; 3 patients, smokers aged > or = 40 years; 4 patients, non-smokers aged > or = 40 years. Nicotine has been tested in 3 different concentrations: 6 microg/ml; 60 microg/ml; 600 microg/ml. To assess cells viability, the neutral red (NR) test was used; to evaluate cell proliferation, the Hoechst test was employed. After 48 h of nicotine exposure, it was found that 600 microg/ml nicotine was strongly cytotoxic to HGF of all groups, with a significant reduction of both proliferation and viability of cells versus control. Comparison between groups of the same age: when comparing untreated HGF (i.e., control values) of smokers < or = 25 years versus non-smokers < or = 25 years, cell proliferation, but not viability, was found to be increased in smokers. Both viability and proliferation of control cells of smokers > or = 40 years were increased versus non-smokers > or = 40 years. HGF of non-smokers < or = 25 years, when exposed to nicotine 600 microg/ml, have less viability and proliferation than HGF of smokers of the same age. Comparison between groups of different age: In the smoker group, untreated HGF (i.e., control values) had similar viability and proliferation, irrespective of age, but nicotine 600 microg/ml kills more HGF in smokers < or = 25 years than in smokers > or = 40 years. In non-smokers, untreated HGF < or = 25 years replicate less, but are not less viable than HGF > or = 40 years. When challenged with nicotine 600 microg/ml, HGF < or = 25 years were less viable than HGF > or = 40 years. From this study, it appears that the smoking history and the patient age could be relevant for final evaluation of the results, since HGF from smokers are less sensitive to nicotine than HGF from non-smokers, and cells from older donors are more resistant to nicotine than cells from younger donors.