To elucidate the roles of cyclin-dependent kinase 6 (cdk6) in T cells, we examined its intracellular localization, kinase activity, and associated proteins in the Jurkat T lymphoblastoid cell line. Jurkat cells had a high level of cdk6, which was associated with cyclin D3, but not cyclin D2, the member of the cyclin D family. When stimulated by a combination of PHA and anti-CD28 mAb, cdk6 activity was up-regulated, as measured by an in vitro kinase assay using recombinant, truncated retinoblastoma tumor suppressor gene protein (Rb protein) as substrate. Activation was most prominent when cells were stimulated with the combination of PHA and anti-CD28, although significant increases were detected after stimulation with PHA alone. The combination also resulted in maximal activation of c-Jun kinase and IL-2 production. Costimulation resulted in a rapid translocation of cdk6 to the nucleus, as demonstrated by both confocal immunofluorescence microscopy and biochemical fractionation techniques. Cdk6 activation and nuclear translocation were also observed after stimulation of Jurkat cells using the anti-CD28 Ab in combination with a mAb to CD3 (OKT3). Furthermore, nuclear translocation was observed in normal human T lymphocytes isolated from peripheral blood and stimulated in vitro with PHA. Two potential endogenous cdk6 substrates (with apparent molecular masses of 75-80 and 55-60 kDa), which were immunoprecipitated with cdk6 and phosphorylated in the in vitro kinase assay, were also identified. These data demonstrate the rapid activation and intracellular translocation of cdk6, implicating this kinase in early signal transduction events in T cells.