3-Nitrotyrosine (NO2Tyr), an L-tyrosine derivative during nitrative stress, can substitute the C-terminus tyrosine of -tubulin post-translationally altering microtubular functions. Since infection of the cells by respiratory syncytial virus (RSV) may require intact microtubules, we tested the hypothesis that NO2Tyr would inhibit RSV infection and intracellular signaling via nitrotyrosination of -tubulin. Human bronchial epithelial cell line (BEAS-2B) was incubated with RSV with or without NO2Tyr. The release of chemokines and viral particles and activation of interferon regulatory factor3 (IRF-3) were measured. Incubation with NO2Tyr increased nitrotyrosinated -tubulin and NO2Tyr co-localized with microtubules. RSV-infected cells released viral particles, RANTES and interleukin-8 (IL-8) in a time- and dose-dependent manner, and intracellular RSV proteins co-precipitated with -tubulin. NO2Tyr attenuated the RSVinduced release of RANTES, IL-8 and viral particles by 50-90% and decreased -tubulin-associated RSV proteins. 3-Chlorotyrosine, another L-tyrosine derivative, had no effects. NO2Tyr also inhibited the RSV-induced shift of the unphosphorylated form I of IRF-3 to the phosphorylated form II. Pre-exposure of the cells to NO₂ (0.15 ppm for 4 hours), which produced diffuse protein tyrosine nitration, did not affect RSV-induced release of RANTES, IL-8 or viral particles. NO2Tyr did not affect the potential of viral spreading to the neighboring cells since the RSV titers were not decreased when the uninfected cells were co-cultured with the pre-infected cells in NO2Tyr-containing medium. These results indicated that NO2Tyr, via replacing the C-terminal tyrosine of -tubulin, attenuated RSV infection and the inhibition appeared to occur at the early stages of RSV infection.