Betel quid (BQ) chewing has a strong correlation with oral leukoplakia, submucous fibrosis and oral cancer. For elucidation of its pathogenesis, we investigated the effects of areca nut (AN) and inflorescence piper betle (IPB) extracts and arecoline on the growth, total DNA synthesis (TDS) and unscheduled DNA synthesis (UDS) of cultured human gingival keratinocytes (GK). Arecoline and AN extract suppressed the growth of GK over 5 days of incubation in a dose-dependent fashion. At concentrations of 100, 200 and 400 μg/ml, AN extract suppressed the growth of GK by 31%, 46% and 90%, respectively. The IPB extracts exerted less inhibitory effect on the growth of GK. IPB extract (200–400 μg/ml) decreased cell numbers by 20–40% over 5 days of incubation. Moreover, at a concentration of 0.1, 0.2 and 0.4 mM, arecoline suppressed cell growth by 44%, 77% and 96%, respectively. However, only AN extract induced TDS and UDS in cultured GK within 6 h of exposure. Induction of UDS by AN extract was concomitant with the presence of apparent intracellular vaculoization. Arecoline was also toxic to GK, but did not induce intracellular vacuolization. At a concentration range of 200–1600 μ/ml, AN extract induced TDS by 2.1- to 6.5-fold. Furthermore, at a concentration of 400–1600 μ/ml, AN extract elevated the UDS by 2.4- to 5.5-fold more than that of untreated control. On the contrary, IPB extract (200–1600 μ/ml) and arecoline (0.2–1.6 mM) inhibited the TDS and UDS of GK to a different extent. Simultaneous exposure of confluent GK to AN extract, IPB extract and arecoline for 1 to 5 days led to different degrees of cytotoxicity that was dose-and time-dependent. These results indicate that AN, IPB and arecoline take part in the pathogenesis of BQ chewing-related oral mucosal lesions, possibly through both genotoxic and non-genotoxic mechanisms.