To determine whether interferon gamma (IFN-gamma) can be used as a biomarker of exposure to viral pathogens, 12-week-old BALB/c mice were injected intraperitoneally with coxsackievirus B3 (CVB3) or coxsackievirus B4 (CVB4) diluted in sterilized phosphate-buffered saline (PBS). Control mice were injected with PBS only. Four months after viral infection, mouse spleen cells were harvested and assayed for the release of IFN-gamma by memory T cells after in vitro stimulation with viral antigens, phytohemagglutinin (PHA), and PBS, respectively. The level of IFN-gamma was examined by an antibody-capture enzyme-linked immunosorbent assay (ELISA). A marked increase in the level of IFN-gamma was observed when memory T cells from CVB3-infected mice were incubated with CVB3 virus, but not with CVB4 or PBS. Conversely, memory T cells from mice infected by CVB4 were not stimulated to produce IFN-gamma when they were incubated with CVB3 and PBS, but did significantly produce IFN-gamma when stimulated with CVB4. T cells from mice injected with PBS did not release IFN-gamma after stimulation with CVB3 or CVB4. However, these T cells did release IFN-gamma after stimulation with PHA. Our results demonstrated that IFN-gamma produced by memory T cells is virus-specific and may have use as a biomarker in viral exposure studies. The results of this study may be extended to the study of infection by pathogens that are capable of inducing cell-mediated immune response in humans.