cAMP-dependent protein kinase (PKA) has been suggested to interfere with T-cell activation by inhibiting interleukin (IL-2) receptor alpha-chain (CD25) expression and IL-2 production. The Ras/MAP kinase pathway has been found to be necessary for induction of the IL-2 production. In this study, we have scrutinized the Ras/MAP kinase pathway in Jurkat T-cells to attempt to identify any sites for PKA-mediated regulatory phosphorylations. Here we unambiguously demonstrate that PKA directly inhibits anti-CD3-induced MAP kinase activation. In vitro phosphorylation experiments showed that Raf-1 was extensively phosphorylated by PKA, while ERK2 and MEK were not. Phosphopeptide mapping identified Ser-43 of Raf-1 as the only site phosphorylated by PKA in the Ras/MAPK pathway. Transient transfection experiments demonstrated that mutations of Ser-43 of the Raf-1 kinase were rendered insensitive to cAMP-mediated inhibition.