Certain protein kinase C (PKC) isoforms, in particular PKCs βII, δ, and ζ, are activated by insulin stimulation. In primary cultures of skeletal muscle, PKCs βII andζ , but not PKCδ, are activated via a phosphatidylinositol 3-kinase (PI3K)-dependent pathway. The purpose of this study was to investigate the possibility that PKCδ may be activated upstream of PI3K by direct interaction with insulin receptor (IR). Experiments were done on primary cultures of newborn rat skeletal muscle, age 5–6 days in vitro. The time course of insulin-induced activation of PKCδ closely paralleled that of IR. Insulin stimulation caused a selective coprecipitation of PKCδ with IR, and these IR immunoprecipitates from insulin-stimulated cells displayed a striking induction of PKC activity due specifically to PKCδ. To examine the involvement of PKCδ in the IR signaling cascade, we used recombinant adenovirus constructs of wild-type (W.T.) or dominant negative (D.N.) PKCδ. Overexpression of W.T.PKCδ induced PKCδ activity and coassociation of PKCδ and IR without addition of insulin. Overexpression of D.N.PKCδ abrogated insulin- induced coassociation of PKCδ and IR. Insulin-induced tyrosine phosphorylation of IR was greatly attenuated in cells overexpressing W.T.PKCδ, whereas in myotubes overexpressing D.N.PKCδ, tyrosine phosphorylation occurred without addition of insulin and was sustained longer than that in control myotubes. In control myotubes IR displayed a low level of serine phosphorylation, which was increased by insulin stimulation. In cells overexpressing W.T.PKCδ, serine phosphorylation was strikingly high under basal conditions and did not increase after insulin stimulation. In contrast, in cells overexpressing D.N.PKCδ, the level of serine phosphorylation was lower than that in nonoverexpressing cells and did not change notably after addition of insulin. Overexpression of W.T.PKCδ caused IR to localize mainly in the internal membrane fractions, and blockade of PKCδ abrogated insulin-induced IR internalization. We conclude that PKCδ is involved in regulation of IR activity and routing, and this regulation may be important in subsequent steps in the IR signaling cascade.