In order to investigate the hypothesis that aberrant expression of cell-cycle regulatory proteins may represent early events in the process of carcinogenesis, levels of expression of the negative regulators p21 waf1/cip1 (p21), p27 kip1 (p27), and p16 ink4a (p16) and/or the positive regulators cyclin D 1 and cyclin E were examined by western blot analysis in cells transformed in vitro by ionizing radiation. The levels of these proteins in 12 independently derived mouse 10T 1 2 cell clones transformed by 1.5 Gy of a radiation were compared with those in nine similarly derived nontransformed control clones. Constitutive levels of p21 were very low in all control clones, whereas p21 expression was significantly elevated in nine of 12 transformed clones. Two of the three transformed clones displaying low levels of p21 expressed increased levels of p53. p21 regulation was also altered in response to radiation in transformed clones as compared with controls, only minimal induction was observed 4 h following g irradiation. Western blot analysis indicated a constant expression of p27 protein but slightly decreased levels of p16 in these transformed clones. Cyclin D 1 was overexpressed in 11 of 12 transformed clones; in only two of these were the levels of cyclin E elevated. Overall, the results suggest that alterations in the expression of cell cycle regulatory proteins may represent important events in radiation-induced oncogenic transformation in vitro. Although the specific alterations vary among different transformed clones, overexpression and aberrant regulation of p21 appear to be the most frequent ones. Mol.