The 90 kDa ribosomal S6 kinases (p90RSKs) are a family of broadly expressed serine/threonine kinases with two kinase domains activated by extracellular signal-regulated protein kinase in response to many growth factors. Our recent study demonstrated that severe acute respiratory syndrome (SARS)-coronavirus (CoV) infection of monkey kidney Vero E6 cells induces phosphorylation and dephosphorylation of signaling pathways, resulting in apoptosis. In the present study, we investigated the phosphorylation status of p90RSK, which is a well-known substrate of these signaling pathways, in SARS-CoV-infected cells. Vero E6 mainly expressed p90RSK1 and showed weak expression of p90RSK2. In the absence of viral infection, Ser221 in the N-terminal kinase domain was phosphorylated constitutively, whereas both Thr573 in the C-terminal kinase domain and Ser380 between the two kinase domains were not phosphorylated in confluent cells. Ser380, which has been reported to be involved in autophosphorylation by activation of the C-terminal kinase domain, was phosphorylated in confluent SARS-CoV-infected cells, and this phosphorylation was inhibited by , which is an inhibitor of p38 mitogen-activated protein kinases (MAPK). Phosphorylation of Thr573 was not upregulated in SARS-CoV-infected cells. Thus, in virus-infected cells, phosphorylation of Thr573 was not necessary to induce phosphorylation of Ser380. On the other hand, Both Thr573 and Ser380 were phosphorylated by treatment with epidermal growth factor (EGF) in the absence of p38 MAPK activation. Ser220 was constitutively phosphorylated despite infection. These results indicated that phosphorylation status of p90RSK by SARS-CoV infection is different from that by stimulation of EGF. This is the first detailed report regarding regulation of p90RSK phosphorylation by virus infection.