Aging and chronic inflammatory syndromes, such as rheumatoid arthritis, are associated with high frequencies of CD4+ CD28 null T cells, which are rarely seen in healthy individuals younger than 40 years. Inasmuch as rheumatoid arthritis and aging are also associated with elevated levels of TNF-α, we examined whether this proinflammatory cytokine influences CD28 expression. Incubation of T cell lines and clones as well as Jurkat cells with TNF-α induced a reduction in the levels of cell surface expression of CD28. This effect of TNF-α was reversible; however, continuous culture of CD4+ CD28+ T cell clones in TNF-α resulted in the appearance of a CD28 null subset. In reporter gene bioassays, TNF-α was found to inhibit the activity of the CD28 minimal promoter. Inactivation of the promoter was accompanied by a marked reduction in DNA-protein complex formation by two DNA sequence motifs corresponding to the transcriptional initiator of the CD28 gene. Indeed, in vitro transcription assays showed that nuclear extracts from TNF-α-treated cells failed to activate transcription of DNA templates under the control of a consensus TATA box and the CD28 initiator sequences. In contrast, similar extracts from unstimulated T cells supported transcription. These results demonstrate that TNF-α directly influences CD28 gene transcription. We propose that the emergence of CD4+ CD28 null T cells in vivo is facilitated by increased production of TNF-α.