The transcription factor NF-kappaB modulates apoptotic machinery following activation by the IkappaB kinase (IKK) complex. Inhibiting activity of one of the catalytic subunits of the IKK complex, IKKbeta (also known as IKBKB and IKK2) severely inhibits NF-kappaB nuclear translocation in response to most stimuli, including ionizing radiation. Doubly floxed Ikkbeta^F/F mice (control) were compared to haplo-insufficient Ikkbeta^F/delta mice (NF-kappaB knockdown) to examine the in vivo apoptotic role of NF-kappaB in the testis. Although Ikkbeta^F/F control adult mice had spermatid head counts and testis and body weights similar to Ikkbeta^F/delta mice, cellular stress in the form of ionizing radiation elicited a differential phenotype. Lower body exposure to 5 Gy of ionizing radiation induced a greater NF-kappaB activation in Ikkbeta^F/F than in Ikkbeta^F/delta mice. In addition, exposure to ionizing radiation resulted in fewer apoptotic germ cells 3, 6, and 12 h after injury in NF-kappaB knockdown mice than in controls, concomitant with the reduced cleavage of caspases 3 and 9 at 3 h. There was also a reduction in total germ cells lost after radiation with NF-kappaB inhibition. Correspondingly, real-time RT-PCR showed a significant reduction in Cdnk1a (also known as p21) and Fasl expression 3 and 6 h, respectively, after irradiation in Ikkbeta^F/delta compared to control testes. These data indicate that, despite acting in an antiapoptotic manner in many tissue types, NF-kappaB is proapoptotic in modulating the germ cell response to ionizing radiation.