Using electrophoretic mobility shift assays (EMSA), we have recently shown that nuclear extracts of 14-day mouse fetal thymocytes contain abundant NF-kappa B transcription factor activity. To determine the functional role of NF-kappa B in early thymocyte development, we have exposed fetal thymus organ cultures to inhibitors of NF-kappa B activation, namely the antioxidants N-acetyl-L-cysteine and butylated hydroxyanisole. Both compounds caused a dose-dependent arrest of thymocyte differentiation toward alpha beta, but not gamma delta, T cells. This was associated with a profound decrease in nuclear content of NF-kappa B and TCF1(alpha) transcription factor activity, as determined by EMSA. In contrast, NF-Y was affected less strongly, and cyclic AMP-response-element-binding protein levels remained essentially unchanged by antioxidants. To test the idea that alpha beta T cell development is correlated with NF-kappa B and TCF1(alpha) activity, we conducted additional experiments in a submersion culture system in which the generation of alpha beta T cells can be manipulated. Standard submersion culture supports gamma delta but alpha beta T cell development. Under these conditions, EMSA showed that transcription factor activities were similar to those seen in the presence of antioxidants. Importantly, when the generation of alpha beta T cells in submersion culture was restored by elevating oxygen concentrations, there was a dramatic increase in TCF1(alpha) activity, and both NF-kappa B and NF-Y returned to control levels. Taken together, these results strongly suggest that NF-kappa B and TCF1(alpha), presumably in concert with other transcription factors, play an important role in the development of alpha beta T cells.