We have investigated the hypothesis that attenuation of the G₂ checkpoint, which delays entry into mitosis in response to damage to DNA and protects against clastogenesis, may contribute to the genetic instability of immortal human cell lines. IMR-90 normal human fibroblasts displayed stringent G₂ checkpoint response to γ-radiation-induced DNA damage. Irradiation with 1.5 Gy induced 98% inhibition of mitosis and 79% inhibition of cyclin B1/p34^CDC2 kinase activity within 2 h. SV40-transformed IMR-90 cells with extended in vitro proliferative lifespan and immortal derivative cells displayed significantly less radiation-induced G₂ delay (60–70%) and less inhibition of cyclin B1/p34^CDC2 protein kinase activity (43–46%) than was seen in normal cells. Two other SV40-transformed lines and a fibrosarcoma line displayed a similar attenuation of G₂ checkpoint function. The attenuation of G₂ checkpoint function in SV40 transformed IMR-90 cells was associated with elevated levels of expression of cyclin B1 (8-fold greater) and p34^CDC2 (2.5-fold greater). By allowing cells with damaged chromatids to enter mitosis, an attenuation of G₂ checkpoint function in finite lifespan cells may promote the genetic alterations necessary for the conversion to immortality.