We have employed in vitro clonogenic assays and DNA flow cytometry to examine the effect of X-ray irradiation on the response of human tumor cells to paclitaxel. Irradiation caused the development of cell cycle delays in G1 and G2/M in the human breast MCF-7 and lung A549 adenocarcinoma cell lines. Irradiation given just prior to or concurrently with exposure to paclitaxel reduced cytotoxicity due to paclitaxel. For example, the surviving fraction of both MCF-7 and A549 cells was 0.03 after 24 h of exposure to 50 nM paclitaxel. However, if the cells were irradiated with 3 Gy just prior to the start of 24 h of paclitaxel exposure, the surviving fractions of MCF-7 and A549 cells were 0.08. Since 3 Gy alone reduced the survival of MCF-7 and A549 cells by 65% and 50%, respectively, the surviving fractions of MCF-7 and A549 cells were 7.6 and 5.3 fold higher after treatment with radiation and paclitaxel than would have been predicted if the cytotoxicities of the different treatments had been additive. Incubation of cells in 5 mM pentoxifylline after irradiation reduced the G2/M block induced by radiation and partially reversed the antagonism of paclitaxel cytotoxicity. These data show that radiation, by inducing cell cycle delays, can antagonize the cytotoxicity of paclitaxel. These results have implications for the development of clinical protocols which combine radiation and paclitaxel in treatment plans.