Methylation of cytosine residues in mammalian DNA is established during gametogenesis and embryogenesis; it plays an important role in gene regulation and normal embryonic development and has also been implicated in genomic imprinting. In the present study, we evaluated whether paternal administration of 5-azacytidine, a drug that is incorporated into DNA and blocks DNA methylation, could alter male germ cell development and function. A drug that does not block methylation, 6-azacytidine, served as a control. Adult male Sprague-Dawley rats (n = 4-8 per group) were treated i.p., three times per week for 4 and 11 wk, with saline or 2.5 (low dosage) or 5.0 (high dosage) mg/kg of 5-azacytidine and 6-azacytidine. After each of the treatment periods, males were mated to determine effects on fertility and embryo development. Although neither 6-azacytidine nor 4 wk of 5-azacytidine treatment affected male reproductive organ weights or sperm counts, 11 wk of 5-azacytidine resulted in dose-dependent reductions in testis and epididymal weights and sperm counts. Both dosages of 5-azacytidine resulted in significant increases in preimplantation loss, and the high dosage of 5-azacytidine caused a decrease in fertility. Examination of embryos on Day 2 of gestation revealed a striking dose-dependent increase in the average number of abnormal embryos per litter sired by the males treated with 5-azacytidine (saline, 0.33 +/- 0.24; low dosage, 2.64 +/- 0.92; high dosage, 10.09 +/- 0.95). In summary, paternal administration of 5-azacytidine interfered with normal male germ cell development and resulted in alterations in fertilization and early embryo development. We suggest that 5-azacytidine-induced alterations in germ cell DNA methylation patterns may be one of the underlying mechanisms, since similar dosages of the analogue 6-azacytidine had no effect on male reproduction and progeny outcome.