We show here that histone deacetylase inhibitors (HDACIs) sodium dibutyrate (SDB) and trichostatin A (TSA) induce a phenotype that has similarities to replicative senescence in human fibroblasts. There was no evidence that SDB accelerated a constitutive cell division counting mechanism as previously suggested because cells pretreated with SDB for three mean population doublings (MPDs) exhibited a similar overall proliferative life span to controls once SDB was withdrawn. SDB-treated cells upregulated the cell cycle inhibitors p21^WAF1 and p16^INK4A, but not p14^ARF, in the same sequential order as in senescence and the cells developed biochemical markers of senescence. However, the mechanism of senescence did not involve telomere dysfunction and there was no evidence for any posttranslational modification of p53. The expression of human papillomavirus (HPV) 16 E6 in human fibroblasts or targeted disruption of the p53 and p21^WAF genes only weakly antagonized HDACI-induced senescence. However, expression of the E7 gene, which inhibits the function of pRb, cooperated with E6 to block SDB-induced senescence completely and human cells deficient in p16^INK4A (but not p14^ARF) were also resistant to SDB-induced senescence, suggesting that the p16^INK4A/pRb pathway is the major mediator of HDACI-induced senescence in human cells. However, p53−/− mouse fibroblasts were resistant to HDACI-induced senescence, identifying p53 as the major pathway to senescence in this species.