Doubly cloned 13762NF rat mammary adenocarcinoma sublines of differing spontaneous metastatic potentials were tested for their response to ionizing radiation in vitro. Cells were irradiated using a 137Cs source, and colony formation was used to measure radiation survival. Heterogeneous responses of the various clones were observed and compared to clonal origin, spontaneous metastatic potential from mammary fat pad sites, and in vitro passage number. Clones MTF4 and MTF7, both derived from the locally growing tumor and possessing intermediate metastatic potentials, exhibited characteristic radiation survival curves (n = 8, D0 = 89 rads, and n = 3, D0 = 78 rads respectively). Clone MTA, also derived from the locally growing tumor but possessing high metastatic potential, had a more linear dose response to ϒ-irradiation (n = 1, D0 = 143 rads, r = -0.99). Clone MTLn2, a lung metastases-derived clone which possesses a low metastatic potential, also exhibited a linear response (n = 1, D0= 175 rads, r = -0.98). At low passage number (T15±1), local tumor-derived clone MTC does not metastasize and possessed higher capacity to accumulate sublethal damage (n = 96, D0 = 150 rads) compared to the other clones, but upon additional subculturing (T21 ±1 clone), MTC became more metastatic and developed an increasingly triphasic dose-response curve, suggesting the existence of at least two cell subpopulations, one which has little capacity to accumulate sublethal damage and the other radioresistant (D0 = 268 rads). Phenotypic drift in ϒ-radiation sensitivity also occurred for the highly metastatic lung metastasis-derived clone MTLn3. At low (T15±1) and high (T43±1) passage, clone MTLn3 was highly metastatic but had different metastatic profiles. Low-passage clone MTLn3 cells possessed a linear dose response (n = 1, D0 = 128 rads, r = -0.99) to ϒ-irradiation; similarly, high-passage MTLn3 cells had a linear dose response but a different inherent sensitivity (n = 1, D0= 152 rads). These studies revealed that considerable clonal heterogeneity exists within a tumor and its metastases with respect to their response to ϒ-radiation. Further, the inherent sensitivities may change with time, thereby altering radiation survival responses. © 1983, American Association for Cancer Research. All rights reserved.