The Tpl‐2 protein serine/threonine kinase was originally identified, in a C‐terminally deleted form, as the product of an oncogene associated with the progression of Moloney murine leukemia virus‐induced T cell lymphomas in rats. The kinase domain of Tpl‐2 is homologous to the Saccharomyces cerevisiae gene product, STE11, which encodes a MAP kinase kinase kinase. This suggested that Tpl‐2 might have a similar activity. Consistent with this hypothesis, immunoprecipitated Tpl‐2 and Tpl‐2deltaC (a C‐terminally truncated mutant) phosphorylated and activated recombinant fusion proteins of the mammalian MAP kinase kinases, MEK‐1 and SEK‐1, in vitro. Furthermore, transfection of Tpl‐2 into COS‐1 cells or Jurkat T cells. markedly activated the MAP kinases, ERK‐1 and SAP kinase (JNK), which are substrates for MEK‐1 and SEK‐1, respectively. Tpl‐2, therefore, is a MAP kinase kinase kinase which can activate two MAP kinase pathways. After Raf and Mos, Tpl‐2 is the third serine/threonine oncoprotein kinase that has been shown to function as a direct activator of MEK‐1.