The possibility of inducing antigen-presenting capacity in cells normally lacking such capacity, currently represents a major goal in vaccine research. To address this issue we attempted to generate 'artificial' APC able to stimulate CD4+ T cell responses when tumor cells were infected with a single, recombinant, vaccinia virus (rVV) containing the two genes encoding murine MHC class II I-Ak and a third gene encoding the murine B7-1 (mB7-1) costimulatory molecule. To minimize the cytopathic effect and to improve safety, in view of possible in vivo applications, we made this rVV replication incompetent by Psoralen and long wave UV treatment. Tumor cells infected with rVV encoding I-Ak alone, pulsed with hen egg white lysozyme peptide (HEL46-61), induced IL-2 secretion by an antigen-specific T hybridoma. Tumor cells infected with the rVV encoding mB7-1 provided costimulation for activating resting CD4+ T cells in the presence of ConA. Tumor cells infected with the rVV encoding I-Ak and mB7-1, and pulsed with chicken ovotransferrin peptide (conalbumin133-145), induced a significantly higher response in a specific Th2 cell clone (D10.G4.1) as compared to cells infected with rVV encoding I-Ak molecules only. Thus, this replication incompetent rVV represents a safe, multiple gene, vector system able to confer in one single infection step effective APC capacity to non-professional APCs.