Tobacco smoking is considered a major risk factor for the development and progression of periodontal diseases (Haber, J. and Wattles, J. (1994). J. Periodontol., 64, 16-23). The purpose of this study was to determine the effects of nicotine on rat gingival fibroblasts (RGF) cultured in vitro. After ether anesthesia, rat gingival tissues were obtained from the attached gingiva of a Wistar rat. Small fragments of gingiva were maintained in culture in Petri dishes. Fibroblasts developing from these explants were collected to obtain monolayer cultures. After the fourth passage (T4), cells were supplemented with nicotine at various concentrations. Control and treated cells were examined under phase contrast or transmission electron microscopy. They were compared as regards their DNA content, mitochondrial activity, collagen and protein synthesis, and cell death by apoptosis or necrosis. Nicotine from 0.05 microM to 1 mM did not affect the DNA content or protein and collagen synthesis. At concentrations between 3 and 5 mM, growth was significantly diminished and the survival rate reduced. Ultrastructural analysis revealed dilated mitochondria and vacuolization in treated cells, suggestive of necrosis, but increased apoptosis was also revealed by cytometry. On the basis of this in vitro study, it appears that tobacco, through its component nicotine, may directly affect various functions of RGF.