Treatment with chemotherapeutics agents may induce persistent DNA damage in male germ cells with the possibility of long-term consequences on fertility and progeny outcome. Telomeres, specialized structures at the physical ends of chromosomes, play an important role in the maintenance of genetic stability and in the response of somatic cells to anticancer drugs. Our objective was to test the hypothesis that exposure to bleomycin, etoposide, or cisplatin (the drugs used to treat testicular cancer) or cyclophosphamide (an anticancer agent and immunosuppressant) targets telomeres in the male germ line. C18-4 spermatogonial cells were exposed to bleomycin, etoposide, cisplatin, or 4-hydroperoxycyclophosphamide (4OOH-CPA, a preactivated analog of cyclophosphamide). All four anticancer drugs induced a significant increase in DNA damage in C18-4 cells, as assessed by gamma-H2AX immunofluorescence. Interestingly, the gamma-H2AX signal was localized to telomeres after treatment with bleomycin, cisplatin, and 4OOH-CPA, but not etoposide. Mean telomere lengths, the intensity of the telomere fluorescence in situ hybridization signal, telomerase activity, and the expression of the telomerase enzyme mRNA components, Tert and Terc, were reduced by exposure to cisplatin and 4OOH-CPA, but not by bleomycin or etoposide. Thus, although all four anticancer drugs induced DNA damage in this spermatogonial cell line, telomeres were not specifically affected by etoposide and only the two alkylating agents, cisplatin and 4OOH-CPA, induced telomere dysfunction. This telomere dysfunction may contribute to infertility and developmental defects in the offspring.