Knowledge about possible genotoxic effects of low-dose radiation on the human germline is limited and relies primarily on extrapolations from high-dose exposures. To test whether ionizing radiation can cause paternal genetic mutations that are transmitted to offspring, we enrolled families of 88 Chernobyl cleanup workers exposed to ionizing radiation. We analyzed DNA isolated from lymphocytes for mutations via DNA blotting with the multi-locus minisatellite probes 33.6 and 33.15 and via PCR in a panel of six tetranucleotide repeats. Children conceived before and children conceived after their father's exposure showed no statistically significant differences in mutation frequencies. We saw an increase in germline microsatellite mutations after radiation exposure that was not statistically significant. We found no dependence of mutation rate on increasing exposure. A novel finding was that the tetranucleotide marker D7S1482 demonstrated germline hypermutability. In conclusion, our results do not support an increased level of germline minisatellite mutations but suggest a modest increase in germline mutations in tetranucleotide repeats. Small sample size, however, limited statistical power.