We used differential display to detect enhanced expression of an mRNA fragment encoding cytokeratin 17 (Ck-17) in respiratory syncytial virus (RSV)-infected epithelial cells. Expression increased 12-fold by 96 h after infection but remained unchanged in cells challenged with virus in the presence of neutralizing anti-RSV fusion protein antibody. Immunoblots of RSV-infected cell lysates probed with an anti-keratin antibody demonstrated stable expression of total cytokeratins over time. When probed with an anti-Ck-17 monoclonal antibody, Ck-17 was first detected at 4 days after infection. In situ staining demonstrated that Ck-17 expression localized to regions of syncytia formation. Expression of Ck-17 mRNA also increased in response to intracellular RSV-F protein in the absence of active RSV infection. No increase in Ck-17 mRNA expression and no syncytia were observed in RSV-infected cells grown in the presence of the NF-kappaB inhibitor gliotoxin. These results suggest that RSV-induced transcriptional activation of the Ck-17 gene is dependent on an NF-kappaB-associated signaling pathway.