In attempt to increase the induction of peptide-specific cytolytic T cells (CTL) we investigated the effect of the Newcastle disease virus (NDV) hemagglutinin-neuraminidase (HN) gene product on the activation of peptide-specific CTL. Spleen cells of CH3 mice immunized against the influenza nucleoprotein peptide 50-63 (NP 50-63) were restimulated in vitro (i) with peptide-pulsed syngeneic fibroblast cells (Ltk-) as antigen-presenting cells, which were in addition (ii) infected with NDV or (iii) stably transfected with the HN cDNA of NDV. A greater than sixfold increase in peptide-specific CTL responses was observed in cultures restimulated with peptide-pulsed Ltk- cells which co-expressed viral hemagglutinin due to either infection or transfection. A similar augmentation was seen in CTL responses against other types of antigen (major histocompatibility complex alloantigens, minor histocompatibility antigens or tumor antigens) when suboptimal cultures were stimulated with the respective antigen-presenting cells modified by NDV infection. These findings suggest that NDV or viral HN expressed on antigen-presenting cells or tumor cells can exert a T cell co-stimulatory function.