Members of the protein kinase C (PKC) family of serine/threonine kinases play key regulatory roles in numerous cellular processes, including differentiation and proliferation. Of the 11 mammalian PKC isoforms known, several have been implicated in tumor development and progression. However, in most cases, isotype specificity is poorly defined, and even contrary functions for a single PKC have been reported mostly because appropriate molecular and genetic tools were missing to specifically assess the contribution of single PKC isoforms in vivo. In this report, we therefore used PKC genetic targeting to study the role of PKCα and PKCζ in colorectal cancer. Both isoforms were found to be strongly down-regulated in intestinal tumors of Apc^Min/+ mice. A deletion of PKCζ did not affect tumorigenesis in this animal model. In contrast, PKCα-deficient Apc^Min/+ mice developed more aggressive tumors and died significantly earlier than their PKCα-proficient littermates. Even without an additional Apc mutation, PKCα knockout mice showed an elevated tendency to develop spontaneous intestinal tumors. Transcriptional profiling revealed a role for this kinase in regulating epidermal growth factor receptor (EGFR) signaling and proposed a synergistic mechanism for EGFR/activator protein and WNT/APC pathways in mediating intestinal tumor development. (Cancer Res 2006; 66(14): 6955-63)