We have previously shown that the nucleocapsid protein of SARS-associated coronavirus (SARS-CoV) activated cyclooxygenase-2 (COX-2) expression (1). In this study, we identified another viral protein, the spike of SARS-CoV, which played an important role in virus-stimulated COX-2 expression after screening all genes from the SARS-CoV genome. We found that an upstream calcium-dependent PKC isozyme PKC that modulates the downstream ERK/NF-B pathway through an influx of extracellular Ca²⁺ is induced by the spike protein of SARS-CoV. The ERK/NF-B was identified to be involved in the activation of COX-2 promoter and production of COX-2 protein in HEK293T cells. We also demonstrated that another unusual pathway, the calcium-independent PI3K/PKC/JNK/CREB pathway, functioned in cooperation with the calcium-dependent pathway to induce COX-2 expression upon stimulation by spike protein. This pathway can be blocked by PKC-specific, small interfering RNA, PI3K/JNK kinase-specific inhibitors as well as dominant negative JNK. PKC-specific siRNA also attenuated the phosphorylation of JNK. Our results provide evidence that helps us understand the function of SRAS-CoV spike protein in SARS pathogenesis.--Liu, M., Yang, Y., Gu, C., Yue, Y., Wu, K. K., Wu, J., Zhu, Y. Spike protein of SARS-CoV stimulates cyclooxygenase-2 expression via both calcium-dependent and calcium-independent protein kinase C pathways.