Four foci (type II or type III) of transformed cells, isolated from the murine line C3H10T1/2 after exposure to proton radiations, were expanded and cytogenetically examined. While the overall numerical chromosome distributions were similar, there were some differences between the various cell lines with regard to the presence and frequency of specific-marker chromosomes and to the colony-forming efficiency in soft-agarose medium. No association between any of these markers and the transformed phenotype could be established. However, in the line F4, derived from a type II focus, numerous double-minute chromosomes (DM) were observed after passage 22, and the phenomenon became more pronounced in the subclone C2. The finding of DMs in radiation-transformed cells is unusual. The DMs were observed in long-term subcultures, and in one of them they were partially replaced by a homogeneously staining chromosome region (HSR). DNAs from transformed cells of the line F4 and subclone C2 was digested with restriction enzymes and analyzed by Southern blotting with probes for seven oncogenes commonly amplified in cancer cells (c-myc, N-myc, N-ras, Ki-ras, Ha-ras, c-myb, c-abl) and with probes for the mouse MHC class I region. None of the regions tested was structurally altered or amplified in these transformed cells. The origin of the genetic material carried by DMs or homogeneously staining intrachromosomal regions (HSR) in cells of the line F4 and subclone C2, where it is believed to provide a selective advantage for in vitro growth, remains unknown.