EMT6 mouse mammary tumor cells were used to examine the cytotoxic effects of 2',2'-difluorodeoxycytidine (gemcitabine; dFdC) alone and in combination with radiation. The cytotoxicity of dFdC differed from that of most antimetabolites. The concentration-response curve for exponentially growing cells treated for 4 hr with various drug concentrations was exponential down to a surviving fraction of 0.05; thus, dFdC appeared to kill cells in all phases of the cell cycle, rather than killing only the S-phase cells that compose approximately 50% of the cell population. High concentrations of dFdC were toxic to cells in plateau phase EMT6 cultures, as well as to cells in rapidly proliferating cultures. These findings are thought to reflect the unusual cellular pharmacokinetics of the compound. Treatment of exponentially growing cultures with dFdC before, during, and after irradiation was extremely effective in killing the cells; the survival curves for cells treated with drug plus radiation were statistically compatible with either additive cytotoxicity or a synergistic effect (i.e., radiosensitization by dFdC). These studies also provided evidence that dFdC released by dying cells could produce delayed cytotoxic effects on cells not treated directly with dFdC. These data provide several bases for expecting beneficial therapeutic effects from antineoplastic regimens combining dFdC plus radiation.