Screening pedigrees for inherited minisatellite length changes provides an efficient means of monitoring repeat DNA instability but has given rise to apparently contradictory results regarding the effects of radiation on the human germline. To explore this further in individuals with known radiation doses and to potentially gain information on the timing of mutation induction, we have used an extremely sensitive single molecule approach to quantify the frequencies of mutation at the hypervariable minisatellites B6.7 and CEB1 in the sperm of three seminoma patients following hemipelvic radiotherapy. Scattered radiation doses to the testicles were monitored and pre-treatment sperm DNA was compared with sperm derived from irradiated pre-meiotic, meiotic and post-meiotic cells. We show no evidence for mutation induction in any of the patients and discuss this finding in the context of previous population studies using minisatellites as reporter systems, one of which provided evidence for radiation-induced germline mutation.