The common leukocyte antigen CD45 plays a central role in T cell activation in coupling the T cell receptor (TCR) to the phosphatidylinositol pathway via interactions with TCR-associated protein tyrosine kinases lck and fyn. We here demonstrate that engagement of CD45 by monoclonal antibodies (mAb) on activated T cells induces tumor necrosis factor (TNF)-alpha as well as TNF-beta, interleukin (IL)-2 and IL-3 gene expression. When human alloreactive T cells are stimulated with mAb 4B2, which recognizes a determinant common to all CD45 isoforms, a vigorous production of TNF-alpha mRNA was detected, which peaked 2 h later. Anti-CD45 mAb cross-linking was required. In contrast, neither mAb 10G10, which recognizes an epitope distinct from the one recognized by mAb 4B2, nor mAb UCHL-1, a CD45RO-specific antibody, induced any significant increase in TNF-alpha transcription. Nuclear run-on transcription assays demonstrated that CD45 cross-linking caused transcriptional activation of the TNF-alpha gene. De novo protein synthesis was not required, since incubation with cycloheximide (CHX) did not block transcriptional activation. CHX in contrast up-regulated TNF-alpha gene expression and increased transcript half-life, an effect that was under control of post-transcriptional mechanisms. Engagement of CD45 by itself did not affect transcript stability. CD45 ligation resulted in TNF-alpha secretion. These results indicate that in addition to its role in TCR/CD3-mediated T cell activation, CD45, in an epitope-specific manner, may act as a primary signaling molecule, leading to the transcriptional regulation and secretion of a major pro-inflammatory cytokine.