Colorectal cancer accounts for approximately 10% of all new cancer cases reported worldwide. High dietary fiber intake has been associated with a reduced risk for this type of neoplasia, and much of this effect is ascribed to the histone acetylase (HDAC) inhibitor n-butyrate produced in the gastrointestinal tract. Natural chemopreventive and several new synthetic HDAC inhibitors exert multiple effects on tumor cells including the induction of differentiation, cell cycle arrest and apoptosis. Since cancer cells undergo mutational changes, it will be important to understand precisely which pathway gains or losses modulate or compromise HDAC inhibitor efficacy. We have recently documented that n-butyrate can provoke apoptosis in human HCT116 colorectal carcinoma cells independently of the p53 tumor suppressor and p21Waf inhibitor. Here, we have developed cell lines on the basis of HCT116 p21-/- cells and HCT116 cells in which the retinoblastoma tumor suppressor protein Rb has been specifically knocked down by antisense expression. The cells were exposed to the DNA-damaging drugs adriamycin (ADR) and etoposide or the HDAC inhibitors n-butyrate and trichostatin A (TSA). While the maximal apoptotic response, observed in the absence of p21Waf, was unaffected by the additional knockdown of Rb when cells were treated with ADR or etoposide, the toxicity of the HDAC inhibitors was significantly reduced. This indicates that hyperphosphorylated Rb itself, dissociated from E2F1 transcription factor, can contribute - directly or indirectly - to tumor cell apoptosis provoked by HDAC inhibitors.