Previous studies of the effects of nicotineon fibroblasts have reported an altered morphology and attachment of fibroblasts to substrates and disturbances in protein synthesis and secretion. This altered functional and attachment response may be associated with changes in the cell membrane resulting from binding of the nicotine, or to disturbances in cell metabolism as a result of high intracellular levels of nicotine. The purpose of the present study, therefore, was to 1) determine whether gingival fibroblasts bound nicotine and if any binding observed was specific or non‐specific in nature; 2) determine whether gingival fibroblasts internalized nicotine, and if so, at what rate; 3) determine whether gingival fibroblasts also released nicotine back into the extracellular environment; and 4) if gingival fibroblasts release nicotine intact or as a metabolite. Cultures of gingival fibroblasts were prepared from gingival connective tissue biopsies. Binding was evaluated at 4° C using a mixture of3H‐nicotine and unlabeled nicotine. Specific binding was calculated as the difference between3H‐nicotine bound in the presence and absence of unlabeled nicotine. The cells bound 1.44 (± 0.42) pmols/106cells in the presence of unlabeled nicotine and 1.66 (± 0.55) pmols/106cells in the absence of unlabeled nicotine. The difference was not significant. Uptake of nicotine was measured at 37° C after treating cells with3H‐nicotine for time periods up to 4 hours. Uptake in pmols/106cells was 4.90 (± 0.34) at 15 minutes, 8.30 (± 0.75) at 30 minutes, 12.28 (± 2.62) at 1 hour and 26.31 (± 1.15) at 4 hours. The efflux or release of nicotine was evaluated after treating cells with3H‐nicotine for 4 hours and then measuring the amount of3H‐nicotine released into fresh non‐radioactive medium over a 2‐hour period by direct scintillation counting of aliquots of medium and by chromatographic analysis of medium samples. Cells released nicotine back into the medium at a rate much slower than the rate of uptake, and the majority of nicotine released by the cells over the 2‐hour period was in the form of nicotine rather than a metabolite. At earlier time points, a greater percentage of the3H isotope separated chromatographically before3H‐nicotine, suggesting an association of the nicotine with intracellular vesicles or other intracellular components. The results of this study suggest that, although nicotine does bind to gingival fibroblasts, this binding is non‐specific, and that the uptake of nicotine by these cells is continuous over 4 hours, providing high intracellular levels. In addition, gingival fibroblasts also release nicotine, apparently unmetabolized, back into the medium. However, an apparent association of the nicotine with intracellular components seems to result in the development of high intracellular levels of nicotine which may interfere with normal cellular functions.J Periodontol 1991; 62:147‐152.