Five monoclonal antibodies (mAbs) specific for porcine interferon-gamma (PoIFN-gamma) were isolated and utilized to develop a PoIFN-gamma sandwich ELISA. Specific reactivity of each mAb with E. coli derived recombinant PoIFN-gamma, but not with rPoIL-2 or rPolL-10, was confirmed in an indirect ELISA and in Western blots. Competitive ELISAs showed that mAbs P2A4 and P2C11 bound an epitope which was not recognized by mAbs P2G10, P1B7 or P2F6. The latter three mAbs were able to neutralize the ability of natural and recombinant PoIFN-gamma to induce the de novo expression of class II MHC antigens on porcine endothelial cells. To simplify the detection of biologically active porcine IFN-gamma, a sandwich ELISA was developed using the mAb P2G10 as a capture antibody and mAb P2C11 as the detecting reagent. The sensitivity of the assay for PolFN-gamma ranged from 1 to 50 ng/ml. Peripheral blood mononuclear cells (PBMC) from all pigs tested produced IFN-gamma when stimulated with either mitogen (PHA) or superantigen (SEB). In contrast, only PBMC obtained from pigs which had previously been vaccinated against PrV produced IFN-gamma in response to stimulation with this virus. Interestingly, cultures with the highest lymphoproliferative response did not necessarily have the highest level of IFN-gamma production.Furthermore, for recall viral antigen, the lymphoproliferative response decreased with time after immunization, whereas the IFN-gamma response increased. Thus, measurement of IFN-gamma production appears to be a good indicator of anti-viral immunological memory.