Mammalian cells at mitosis, differentiated lymphocytes, and some radiation-hypersensitive mutants in interphase contain all or a measurable portion of their chromatin in condensed/compacted form and are hypersensitive to ionizing radiation by the mechanism described by single-hit inactivation kinetics (alpha). These observations led to the investigation as to whether compacted chromatin in interphase is the target that determines the widely variable alpha-parameters and surviving fractions of 2 Gy (SF2Gy) measured for human tumor cell lines. Six cell lines whose SF2Gy ranged from 0.29 to 0.73 were used for this study. Their different radiosensitivities were associated mainly with differences in their single-hit inactivation parameters (alpha). Electron microscope images of interphase nuclei were optically scanned, and the pixel densities were digitized for quantitative analyses. A significant correlation between the percentage of nuclear pixels with densities similar to those found in mitotic chromosomes (percent compacted chromatin) and the alpha-inactivation parameters was observed. Digital analyses of electron and/or confocal microscope images of chromatin in interphase tumor cells in biopsy specimens could become a rapid assay for predicting the intrinsic radiosensitivity of tumor clonogens. This research has also identified some inhibitors of protein (histone) phosphatases that promote chromatin compaction and radiosensitize cells to 2-Gy dose fractions.