The addition of benzo(a)pyrene to cells obtained from Syrian hamster embryo cultures that had been plated to produce discrete colonies resulted in transformation. Altered colonies were not seen in controls. Prior irradiation of hamster embryo cells increased the transformation by benzo(a)pyrene, both per cell and per total colony. Maximum enhancement occurred when benzo(a)pyrene was added 48 hr after irradiation. Enhancement was greatest after 250-500 R, and decreased with higher doses of radiation. Although the link between radiation (both x-and UVirradiation) and cancer has been recognized since the discoveries of x-rays and natural radioactivity, the use of physical agents as carcinogens or cocarcinogens for in vitro studies has been extremely limited. X-irradiation has been shown to promote in vitro transformation in combination with polyoma (4) or simian virus 40 (5, 6) in hamster embryonic secondary cultures, hamster cell line BHK 21/13, and 3T3 mouse cell lines. Evidence was presented that the effect in established cell lines was not due to selection of preexisting cells with a greater susceptibility to transformation. Cell transformation in vitro by x-irradiation only has been reported in primary cell lines of hamster embryo cells (7), but has not been observed in the irradiated controls of the experiments involving radiation and viruses or after repeated weekly irradiation of a hypotetraploid fibroblast line derived from mouse spleen (8).Abbreviation: BP, benzo(a)pyrene. MATERIALS AND METHODSThe tissue-culture fluids and medium as well as the feeder layer cultures, the source and culture of cells from minced hamster embryos, and the standard conditions for the transformation assay have been described in detail (2). Cells derived from whole rat or hamster fetuses were grown as monolayers in Dulbecco's modification of Eagle's medium (MEM) with either 10% calf serum or 10% fetal bovine serum. In all experiments 2-to 4-day-old secondary or tertiary hamster cultures obtained by seeding 5 X 106 cells/100 ml plastic dish were used. Feeder layers composed of secondary cultures of rat embryo cells were irradiated as confluent monolayer cultures in 2 ml of medium. A Picker Portable Industrial x-ray apparatus (T55-433) was used at 110 kV and 5 mA, 2225 R/min, adjusted so that a total of 5000 R in air was delivered to a distance of 22.2 cm. Hamster cells were irradiated as confluent monolayers in 100-mm plastic Petri dishes with a Westinghouse Quandrocondex machine with two tubes, above and below, with the following factors: 200 kV (CP), 0.25 Cu + 0.55 mm Al filtration in each tube (HVL 0.9 mm Cu), 15 mA, 54 cm TSD, output 139 R/min. After being irradiated with 150-1000 R, the cells were plated on irradiated rat fibroblast feeder layers in 60-mm Petri dishes. Each value represents data obtained from at least 12 plates. Because unirradiated and irradiated cells had different survival rates, it was necessary to vary the number of hamster cells seeded to obtain the same approximate number of colonies. At desig...