A specific antigenic ligand and a costimulatory signal are required for optimal T cell activation. We constructed and tested recombinant Vaccinia viruses (recVV) expressing hB7-1 or hB7-2 as a gene expression vector system for delivery of costimulatory function in vitro. Upon infection with replication incompetent and non-cytopathic recVV B7, all human tumour cell lines tested expressed the recombinant molecules. Cell lines expressing recombinant B7 molecules provided effective costimulation for proliferation of resting CD4+ T helper cells in the presence of suboptimal PMA concentrations. The costimulatory effect could be blocked with soluble CTLA-4 proteins. RecVV B7-1 infected EBV transformed B-lymphocytes overexpressed the costimulatory molecules resulting in enhanced costimulation. The capacity of these cells to stimulate autologous CD4+ memory cells of VV immunocompetent donors was not impared by the recVV infection indicating an intact capacity for processing and presenting antigenic proteins in the context with MHC Class II molecules remained. RecVV encoding human B7 molecules therefore appear to be promising experimental and clinical tools to enhance immune responses.