Abstract The viral antigens recognized by cytotoxic T cells (CTL) have not been defined in most viruses infecting mouse or man. Natural or artificial virus recombinants can be used to determine the antigen specificity of CTL directed against viruses with segmented genomes, such as influenza, but this technique is more difficult to apply to the study of unsegmented viruses. We describe here the use of recombinant vaccinia viruses, containing cDNA corresponding to either the nucleoprotein (N) gene or the major surface glycoprotein (G) gene of human respiratory syncytial virus (RSV), to examine the antigen specificity of anti-RSV cytotoxic T cells from humans and mice. The results demonstrate that the RSV N protein is one of the target antigens for CTL in man and mouse, whereas the G protein was not recognized and can at best represent a minor target antigen for CTL.