Stimulation of T cells with antibodies directed towards the T cell receptor complex results in the activation of mitogen-associated protein kinase (MAPK). Two pathways have been described in other cell types that can lead to MAPK activation. One of these pathways involves the activation of Ras, leading to the activation of Raf-1, and the subsequent activation of MEK (MAPK or ERK kinase). The contribution of this pathway in T cells for anti-CD3 or phorbol myristate acetate (PMA)-mediated MAPK activation was examined. We detected the kinase activities of Raf-1 and MEK towards their substrates (MEK for Raf-1 and MAPK for MEK) in this pathway leading to the activation of MAPK. Stimulation of the T cells with either anti-CD3 antibody or PMA resulted in a rapid activation of both Ras and Raf-1. MEK activity towards kinase-active or -inactive recombinant MAPK also increased upon stimulation. In addition, both MAPK and p90rsk were activated in these cells. We suggest that activation of MAPK and the subsequent activation of ribosomal S6 kinase (p90rsk) occurs by the Ras/Raf-1/MEK cascade in T lymphocytes stimulated by ligation of the T cell receptor complex.