Epidermal growth factor (EGF) has been reported to act as a tumor promoter in several tissues, such as skin, in association with the induction of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). However, molecular mechanisms involved in these regulations are not well defined. This study addressed a potential role of nuclear factor of activated T cells 3 (NFAT3) in EGF-induced COX-2 and iNOS transcription and cell transformation in mouse epidermal Cl 41 cells. We found that EGF markedly induced anchorage-independent growth (cell transformation) of Cl 41 cells, as well as COX-2 (> 6-fold) and iNOS (> 5-fold) promoter-dependent transcription. The EGF-induced COX-2 transcription was blocked by knockdown of NFAT3 with NFAT3 siRNA, whereas the transcription of iNOS and cell transformation induced by EGF were not affected. Although our recent studies supported that NFAT3 plays an essential role in chemical carcinogen benzo[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE)-induced cell transformation, the data presented here demonstrated that NFAT3 is required for EGF-induced COX-2 transcription, but neither iNOS transcription nor cell transformation, indicating that the role of NFAT3 in regulating cell transformation is carcinogen-specific.