NF-kappaB is a transcription factor that regulates a variety of genes involved in the control of the immune and inflammatory responses. Activation of NF-kappaB is mediated by an inducible I-kappaB kinase (IKK) complex comprised of two catalytic subunits, IKKalpha and IKKbeta. In this study, the role of these kinases in the development and function of T lymphocytes was explored using transgenic mice expressing the dominant-negative forms of one or both kinases under the control of a T cell-specific promoter. Activation of the NF-kappaB pathway in thymocytes isolated from these transgenic mice following treatment with either PMA and ionomycin or anti-CD3 was markedly inhibited. Although inhibition of IKKalpha and/or IKKbeta function did not alter T cell development in these transgenic mice, the proliferative response to anti-CD3 was reduced in thymocytes isolated from mice expressing dominant-negative IKKbeta. However, inhibition of both IKKalpha and IKKbeta was required to markedly reduce cytokine production in thymocytes isolated from these transgenic mice. Finally, we demonstrated that IKKalpha and IKKbeta have opposite roles on the regulation of anti-CD3-induced apoptosis of double-positive thymocytes. These results suggest that IKKalpha and IKKbeta have distinct roles in regulating thymocyte function.