Apoptosis is an important cellular response to UV radiation (UVR), but the corresponding mechanisms remain largely unknown. Here we report that the p85alpha regulatory subunit of phosphatidylinositol 3-kinase (PI-3K) exerted a proapoptotic role in response to UVR through the induction of tumor necrosis factor alpha (TNF-alpha) gene expression. This special effect of p85alpha was unrelated to the PI-3K-dependent signaling pathway. Further evidence demonstrated that the inducible transcription factor NFAT3 was the major downstream target of p85alpha for the mediation of UVR-induced apoptosis and TNF-alpha gene transcription. p85alpha regulated UVR-induced NFAT3 activation by modulation of its nuclear translocation and DNA binding and the relevant transcriptional activities. Gel shift assays and site-directed mutagenesis allowed the identification of two regions in the TNF-alpha gene promoter that served as the NFAT3 recognition sequences. Chromatin immunoprecipitation assays further confirmed that the recruitment of NFAT3 to the endogenous TNF-alpha promoter was regulated by p85alpha upon UVR exposure. Finally, the knockdown of the NFAT3 level by its specific small interfering RNA decreased UVR-induced TNF-alpha gene transcription and cell apoptosis. The knockdown of endogenous p85alpha blocked NFAT activity and TNF-alpha gene transcription, as well as cell apoptosis. Thus, we demonstrated p85alpha-associated but PI-3K-independent cell death in response to UVR and identified a novel p85alpha/NFAT3/TNF-alpha signaling pathway for the mediation of cellular apoptotic responses under certain stress conditions such as UVR.