We have examined the effects of commonly used chemotherapeutic agents on human colon cancer cell lines in which the p53 pathway has been specifically disrupted by targeted homologous recombination. We found that p53 had profound effects on drug responses, and these effects varied dramatically depending on the drug. The p53-deficient cells were sensitized to the effects of DNA-damaging agents as a result of the failure to induce expression of the cyclin-dependent kinase inhibitor p21. In contrast, p53 disruption rendered cells strikingly resistant to the effects of the antimetabolite 5-fluorouracil (5-FU), the mainstay of adjuvant therapy for colorectal cancer. The effects on 5-FU sensitivity were observed both in vitro and in vivo, were independent of p21, and appeared to be the result of perturbations in RNA, rather than DNA, metabolism. These results have significant implications for future efforts to maximize therapeutic efficacy in patients with defined genetic alterations.