This study identifies memory cytotoxic T lymphocyte (CTL) epitopes to respiratory syncytial virus (RSV) in healthy South African adults and demonstrates the conservation of those epitopes in circulating field strains of RSV in South Africa. Thirty-seven healthy adults from a population with diverse HLA backgrounds were screened by gamma interferon (IFN-gamma) enzyme-linked immunospot for memory CTL activity in response to overlapping peptides representing the complete nucleoprotein (N) of RSV. Responses of more than 40 spot-forming cells/million cells were detectable in 21 individuals. The significant responses were further characterized, and 14-mer peptides were identified that induced cytolytic activity. Fine mapping of peptides with the highest cytolytic activity identified an HLA-B(*)08-restricted RSV-specific CTL epitope. The extended 14-mer peptide containing this epitope also induced lysis in the context of A(*)02-restricted target cells in some individuals. These HLA types are common in the target population; thus, the epitope is useful for studies of CTL responses to RSV in humans. The epitope was detected in healthy adults, reflecting the response generated in the course of previous natural RSV infection. We obtained a large panel of naturally occurring isolates of RSV to determine whether there was evidence of escape from CTL activity in circulating strains. We found that this epitope and a previously identified B(*)07-restricted N protein epitope were conserved in RSV field strains representing the diversity of circulating genotypes. This work suggests that escape from CTL activity is not common for this acute respiratory infection.