Ionizing radiation can induce genomic instability in the progeny of irradiated cells, as was demonstrated in various experimental systems. Most in vitro studies have utilized replicating cells, but it is not clear whether radiation-induced genomic instability persists in quiescent cells. Here we show the induction of X-ray-induced genomic instability in normal human diploid cells irradiated and maintained in a quiescent state for up to 24 months while cells were subcultured approximately once every 2-3 months. Every 12 months, a fraction of the irradiated cell population was stimulated to divide by culturing at a low density, and we found that these cells showed increased frequencies of phosphorylated ATM foci, decreased colony-forming ability, and increased frequency of chromosomal aberrations. No significant increases in ROS levels were detected in long-term cultured cells. These results suggest that there are ROS-independent mechanism(s) induced by radiation, which can generate persistent delayed effects in quiescent cells, and could ultimately contribute to carcinogenesis.