p70 S6 kinase (p70 S6K) has been implicated in the regulation of cell cycle progression. However, the mechanism of its activation is not fully understood. In the present work, evidence is provided that an atypical protein kinase C (PKC) isotype, PKCλ, is indispensable, but not sufficient, for the activation of p70 S6K. Both the regulatory and kinase domains of PKCλ associate directly with p70 S6K. Overexpression of the kinase domain without kinase activity or the regulatory domain of PKCλ results in the suppression of the serum-induced activation of p70 S6K. In addition, two types of dominant-negative mutants of PKCλ, as well as a kinase-deficient mutant of p70 S6K, suppress serum-induced DNA synthesis and E2F activation. The overexpresion of the active form of PKCλ, however, fails to activate p70 S6K. These results suggest that PKCλ is a mediator in the regulation of p70 S6K activity and plays an important role in cell cycle progression.