The proto-oncogene Cot/Tpl-2 encodes a MAP3K-related serine-threonine kinase. Expression of wild type Cot activates the IkappaB kinases (IKK) leading to induction of NF-kappaB. Conversely, expression of kinase-deficient Cot inhibits CD3/CD28 but not TNF alpha induction of NF-kappaB. These findings suggest the selective involvement of Cot/Tpl-2 or a closely related kinase in the CD3/CD28 costimulatory pathway leading to induced nuclear expression of NF-kappaB. In contrast, a kinase-deficient mutant of the NF-kappaB-inducing kinase (NIK) inhibits both CD3/CD28 and TNF alpha signaling, indicating that these pathways converge at or prior to the action of NIK. Consistent with such a sequential function of these two kinases, Cot physically assembles with and phosphorylates NIK in vivo.