To define the in vivo role of individual PKC isoforms in mouse skin carcinogenesis, we previously characterized FVB/n transgenic mice that over‐expressed epitope‐tagged PKCδ (T7‐PKCδ) or PKCϵ (T7‐PKCϵ) isoforms under the regulation of the human K14 promoter. In continuation of our prior PKC isoform specificity studies, we now report the generation of FVB/n transgenic mice with K14‐regulated, epitope‐tagged PKCα (T7‐PKCα). T7‐PKCα transgenic mice (line 115) express 8‐fold more PKCα protein than wild‐type mice. Using high‐resolution immunogold cytochemistry, we determined that transgenic over‐expression of T7‐PKCα did not alter the subcellular localization of PKCα but that the density of PKCα staining increased. PKCα localized primarily to the cytoskeleton (tonofilaments, tight junctions) and cell membranes, with modest but definite nuclear labeling also identified. Also, PKCα over‐expression did not alter the immunoreactive protein levels of other PKC isoforms (δ, ϵ, η, ζ, μ) in the epidermis. Skin tumor‐promotion susceptibility was compared among all 3 lines of T7‐PKC transgenic mice (α, δ and ϵ). While T7‐PKCα had no effect on skin tumor promotion by TPA, T7‐PKCδ reduced papilloma burden by 76% compared to wild‐type controls. T7‐PKCϵ further reduced papilloma burden to 93% compared to wild‐type controls but still resulted in the development of squamous‐cell carcinoma. To find potential mechanisms of PKC‐associated differences in tumor promotion, the induction of known downstream effectors of tumor promotion, ornithine decarboxylase (ODC) activity and epidermal hyperplasia, was determined. Despite long‐term papilloma inhibition in both PKCδ and PKCϵ transgenic mice, the induction of ODC by TPA was not attenuated in PKC δ and ϵ mouse lines. Both PKC transgenic and wild‐type mice exhibited sustained hyperplasia after repeated TPA treatments. However, TPA‐induced epidermal hyperplasia in T7‐PKCϵ mice was significantly increased (52%) compared with T7‐PKCα, T7‐PKCδ and wild‐type mice. TPA‐induced ODC activity and the resultant accumulation of polyamines may play different roles (e.g., induction of apoptosis vs. proliferation) in the pathways leading to the induction of cancer in PKCα, PKCδ and PKCϵ transgenic mice. © 2001 Wiley‐Liss, Inc.