To examine the role of NF-kappaB in T cell development, we analyzed thymocyte ontogeny in transgenic (mutant I-kappaBalpha (mI-kappaBalpha)) mice that express a superinhibitory form of the NF-kappaB inhibitory protein, I-kappaBalpha (I-kappaBalpha(A32/36)), under the control of the T cell-specific CD2 promoter and enhancer. Thymi from mI-kappaBalpha mice contained increased numbers of double-positive (DP) and decreased numbers of both CD4(+) and CD8(+) single-positive cells, consistent with a block in DP thymocyte maturation. In addition, expression of CD69, a marker of positive selection, was decreased on DP thymocytes from the mI-kappaBalpha mice. To test directly whether NF-kappaB was required for positive or negative selection, we generated mI-kappaBalpha mice expressing the H-Y or 2C alphabeta TCR transgenes. Expression of the I-kappaBalpha(A32/36) transgene caused a block in the positive selection of CD8(+) single-positive cells in both strains of TCR transgenic animals. In contrast, negative selection was unaffected by expression of the I-kappaBalpha(A32/36) transgene. Taken together, these results identified a NF-kappaB-dependent transcriptional pathway that is selectively required for the positive selection of CD8(+) thymocytes.