Abstract Neutralization by rabbit antisera to Murray Valley encephalitis, West Nile, Kunjin, and Edge Hill viruses was studied by plaque assay of surviving virus. The neutralization slope of the regression lines in dose-response experiments for each hyperimmune serum was significantly greater in homologous reactions than in cross reactions. The regression line equations were transformed to an “area function” which represented a summation of the neutralizing activity over the whole range of antiserum dilutions, providing a useful basis for comparing the potency of antisera. Normalizing these area functions permitted evaluation of antigenic relationships. The inclusion of fresh serum in the tests increased neutralization in most cases, but specificity was not improved. In contrast to the results of neutralization tests determined by intraperitoneal inoculation in mice, hyperimmune sera exhibited greater specificity than immune sera in the plaque assay, and in the plaque inhibition test. Kunjin antisera displayed similar neutralizing activity against both Kunjin and West Nile viruses in all three assay systems. Values for the neutralization slopes were considered in relation to an affinity term for interaction between small molecules and protein. Variations in slope with the same serum assayed in different hosts or against different viruses were interpreted as being due to recognition by the specific host cell of different dispositions of antigen-antibody complexes comprising a critical area on each neutralized virus particle.