CTL play a major role in the clearance of respiratory syncytial virus (RSV) during experimental pulmonary infection. The fusion (F) glycoprotein of RSV is a protective Ag that elicits CTL and Ab response against RSV infection in BALB/c mice. We used the strategy of screening a panel of overlapping synthetic peptides corresponding to the RSV F protein and identified an immunodominant H-2Kd-restricted epitope (F85-93; KYKNAVTEL) recognized by CD8+ T cells from BALB/c mice. We enumerated the F-specific CD8+ T cell response in the lungs of infected mice by flow cytometry using tetramer staining and intracellular cytokine synthesis. During primary infection, F85-93-specific effector CD8+ T cells constitute ∼4.8% of pulmonary CD8+ T cells at the peak of the primary response (day 8), whereas matrix 2-specific CD8+ T cells constituted ∼50% of the responding CD8+ T cell population in the lungs. When RSV F-immune mice undergo a challenge RSV infection, the F-specific CD8+ T cell response is accelerated and dominates, whereas the primary response to the matrix 2 epitope in the lungs is reduced by ∼20-fold. In addition, we found that activated F-specific effector CD8+ T cells isolated from the lungs of RSV-infected mice exhibited a lower than expected frequency of IFN-γ-producing CD8+ T cells and were significantly impaired in ex vivo cytolytic activity compared with competent F-specific effector CD8+ T cells generated in vitro. The significance of these results for the regulation of the CD8+ T cell response to RSV is discussed.