A modified antibody-forming cell assay was used to enumerate splenocytes secreting antibodies to Japanese encephalitis (JE) virus and four other flaviviruses in infected cells as the target antigens. The optimal viral antigen expression for the assay was standardized in the infected cells for each virus and the incubation time varied depending of the cell line and the virus. The kinetics of response of JE virus-infected mice readily showed IgG isotype switching. Antibody-forming splenocytes of mice primed or boosted with one flavivirus could distinguish, in variable proportions, the homologous virus antigen from heterologous ones depending on the serocomplex of the virus. Antibody-forming cells from flavivirus-infected mice were flavivirus specific as evidenced by the lack of recognition of Getah virus (alphavirus) antigen. After JE virus-infected mice were cross-primed with another flavivirus, the IgG-forming cell response resembled the secondary response to both viruses but had higher affinity to JE virus than to the cross-priming virus.