Stimulation of the T cell antigen receptor (TCR) induces a number of intracellular signaling pathways which lead to the transcription of a variety of new genes. Of the newly synthesized proteins, the earliest to be detected on the cell surface is the type II integral membrane protein CD69. Cross-linking of this activation antigen induces signaling events related to T cell activation. The proto-oncogene product Ras has been reported to up-regulate CD69. However, which of the potential effectors of Ras induces the expression of CD69 has remained unclear. Using transient transfection, we have shown a constitutively active form of the serine/threonine kinase Raf-1 to be sufficient to induce CD69 expression in human Jurkat T cells. Raf-1 was further shown to be necessary for PMA-induced CD69 expression, since transfection of a dominant inhibitory form of Raf-1 blocked the up-regulation of CD69 by PMA. In addition, studies with the calcium ionophore ionomycin identified a previously uncharacterized pathway regulating the expression of CD69 in T cells. Elevation of intracellular calcium induced the expression of CD69 in both Jurkat cells and peripheral blood T cells. This effect was sensitive to the immunosuppressive drug cyclosporin A, indicating that calcium-induced CD69 expression is mediated by the protein phosphatase calcineurin. Taken together, these results define Raf-1 as the major signaling mediator of CD69 expression in T cells and suggest that multiple mechanisms exist to regulate the level of CD69 expression following TCR stimulation.