The preferential differentiation of T helper (Th) cells to Th1 or Th2 subsets is important with respect to susceptibility or resistance to particular infections, or to autoimmune diseases and allergic diseases. To more effectively drive immune responses toward antigen-specific Th1 responses, we constructed a mammalian expression vector (pOVA/IFN-gamma) carrying a hybrid gene in which the ovalbumin (OVA) (a model antigen) cDNA was covalently linked to murine interferon-gamma (IFN-gamma) cDNA. Intramuscular injection of BALB/c mice with the pOVA/IFN-gamma DNA increased both the production of OVA-specific IFN-gamma by CD4+ T cells and the ratio of anti-OVA immunoglobulin G (IgG) 2a to IgG1 isotypes, while the injection with the pOVA alone, or with the mixture of the pOVA and pIFN-gamma, caused no or little increase. Furthermore, the OVA-specific, Th1 immune responses were dramatically augmented by multiple injections with the pOVA/IFN-gamma DNA. These studies indicate that the direct linkage of an OVA gene to an IFN-gamma gene in the expression plasmid is required for efficiently confining the Th1 effects of IFN-gamma to the OVA-specific cells, and the linkage effect of the OVA/IFN-gamma DNA can be potentiated by multiple vaccination.