The cellular response to chemoradiotherapy was investigated in cells of the HCT116 (wild-type TP53) and HT29 (mutated TP53) human colorectal cancer cell lines to better understand how the chemotherapeutic agent 5-fluorouracil (5-FU) acts as a radiosensitizer in vitro and how it contributes to the well-documented greater efficacy of chemoradiotherapy compared to radiotherapy (or chemotherapy) alone. A bolus 5-FU treatment protocol that simulated actual clinical clearance kinetics was used with a radiation dose given within 90 min after drug addition. The involvements of key signaling pathways (DNA damage response, cell cycle progression, cell proliferation, cell death) in cell responses were investigated concurrently, allowing for direct correlations of numerous treatment response phenotypes. Early DNA damage response, substantial cell death, loss of clonogenicity, and senescence characterized both radiotherapy- and chemoradiotherapy-treated cultures but not chemotherapy-treated cultures. The largest G(2)/M arrests and strongest correlation of senescence with non-clonogenicity were seen in radiotherapy- and chemoradiotherapy-treated HCT116 cell cultures, suggesting that functional TP53 could play a role in maintaining/inducing these cellular phenotypes. Overall, chemoradiotherapy proved to be the most effective treatment modality since it resulted in the strongest growth inhibitions, largest G(2)/M arrests, largest fractions of senescent cells, and complete loss of clonogenicity in both cell lines.