Protein kinase C (PKC), a family of lipid-activated serine kinases, is involved in multiple functions in the regulation of growth control. The PKC-related isoform PKC mu/PKD has been implicated in mitogenic signal cascades because of the activation of p42/p44 MAPK leading to Elk1-mediated gene transcription, and PKC mu/PKD has been shown to be activated via a PKC-dependent pathway. By using confocal analyses, we demonstrate here that PKC mu partially colocalizes with PKC eta in different cell types. Colocalization depends on the presence of the PKC mu pleckstrin homology domain. Coexpression of constitutively active PKC eta with PKC mu leads to a significant enhancement of the PKC mu substrate phosphorylation capacity as a result of an increased phosphorylation of the activation loop Ser(738/742) of PKC mu, whereas Ser(910) autophosphorylation remains unaffected. In vitro phosphorylation experiments show that PKC eta directly phosphorylates PKC mu on activation loop serines. Consequently, the p42 MAPK cascade is triggered leading to an increase in reporter gene activity driven by a serum-responsive element in HEK293 cells. At the same time, PKC eta-mediated JNK activation is reduced, providing evidence for a mutual regulation of PKC mu/PKC eta affecting different arms of the p38/ERK/JNK pathways. Our data provide evidence for the sequential involvement of selective PKC isoforms in kinase cascades and identify the relevant domains in PKC mu for interaction with and activation by PKC eta as pleckstrin homology domain and activation loop.