Photodynamic Therapy (PDT) is an approved anticancer therapy that kills cancer cells by the photochemical generation of reactive oxygen species following absorption of visible light by a photosensitizer, which selectively accumulates in tumors. We report that hypericin-mediated PDT of human cancer cells leads to up-regulation of the inducible cyclooxygenase-2 (COX-2) enzyme and the subsequent release of PGE2. Dissection of the signaling pathways involved revealed that the selective activation of p38 MAPK alpha and beta mediate COX-2 up-regulation at the protein and messenger levels. The p38 MAPK inhibitor, PD169316, abrogated COX-2 expression in PDT-treated cells, whereas overexpression of the drug-resistant PD169316-insensitive p38 MAPK alpha and beta isoforms restored COX-2 levels in the presence of the kinase inhibitor. Transcriptional regulation by nuclear factor-kappaB was not involved in COX-2 up-regulation by PDT. The half-life of the COX-2 messenger was drastically shortened by p38 MAPK inhibition in transcriptionally arrested cells, suggesting that p38 MAPK mainly acts by stabilizing the COX-2 transcript. Overexpression of WT-p38 MAPK increased cellular resistance to PDT-induced apoptosis, and inhibiting this pathway exacerbated cell death and prevented PGE2 secretion. Hence, the combination of PDT with pyridinyl imidazole inhibitors of p38 MAPK may improve the therapeutic efficacy of PDT by blocking COX-2 up-regulation, which contributes to tumor growth by the release of growth- and pro-angiogenic factors, as well as by sensitizing cancer cells to apoptosis.