Ionizing radiation is a known carcinogen and teratogen. However, the point mutations produced by ionizing radiation in mammalian cells have not been fully characterized. Determination of a characteristic spectrum of X-ray-induced mutations in mammalian cells could provide clues to cellular repair processes and could serve as a marker of individual exposure to radiation. Mouse fibroblasts containing in their genome multiple copies of a recoverable lambda phage shuttle vector were used to detect and analyze radiation-induced point mutations in the supF mutation reporter gene. Following fractionated doses of ionizing radiation, a unique mutational spectrum notable for a high proportion of T:A-->G:C transversions (57%) was found. This pattern was distinct from the spectra of UV-induced and spontaneous mutations detected in the same mouse cell assay system (mainly C:G-->T:A transitions). The predominance of T:A-->G:C transversions and the pattern of mutation hot-spots are consistent with a possible role for polymerase beta in the repair of X-ray-damaged DNA. These results may also help to define a distinctive mutational signature of X-ray exposure in mammalian cells.