Retinoic acid (RA) plays an important role in the regulation of cell growth and differentiation. To investigate whether RA extends in vitro the life span of human epithelial cells, we examined the effect of all-trans RA on both the cumulative population-doubling level (PDL) and the replicative senescence of cultured oral keratinocytes. When proliferating oral keratinocytes were cultured in medium containing 1 nM of all-trans RA, the in vitro life span of the cells was increased 1.5- to 1.8-fold compared to the vehicle control and the replicative senescence of the cells was significantly inhibited. Since the replicative senescence of human epithelial cells is associated with a steady increase of p16(INK4A) and a loss of telomerase activity, we expected that RA could delay the replicative senescence of oral keratinocytes by decreasing p16(INK4A) expression and/or inhibiting the loss of telomerase activity. To test this possibility, we examined the expression of replicative senescence-associated genes and the telomerase activities of different PDL numbers of oral keratinocytes exposed to 1 nM of all-trans RA. The protein level of cellular p16(INK4A) in the RA-treated oral keratinocytes was gradually but significantly enhanced by an increased PDL number; however, the level was significantly lower than that of the vehicle control at all of the same PDL numbers. In contrast, the telomerase activity was maintained in oral keratinocytes with increasing PDL numbers induced by RA treatment. Summarizing, these results indicate that RA induces the in vitro life-span extension of oral keratinocytes, which is linked to a decreased cellular level of p16(INK4A) and the maintenance of telomerase activity.