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Objective To investigate the role of Anopheles subpictus Grassi as a vector of Japanese encephalitis virus (JEV) transmission in Cuddalore, an area of Tamil Nadu endemic for the disease. Method We collected 98 pools (4900 specimens) of wild adult male An. subpictus mosquitoes outdoors during dusk hours and screened them for JEV antigen by antigen-capture Enzyme Linked Immunosorbent Assay. Additionally, over a period of 1 year, we tested 166 pools (8300 specimens) of wild adult female An. subpictus mosquitoes collected indoors for JEV. Results Four pools of male An. subpictus tested positive. This indicates possible natural transovarial transmission of the virus through An. subpictus. Nineteen female pools were positive with a minimum infection rate of 2.3. From January through March the maximum infection rate was highest: 5.0 compared with 1.7 between April and September and 2.1 from October to December, although the difference was not statistically significant. From the 19 positive female pools, four isolates were confirmed as JEV by insect bioassay. Conclusion The role of An. subpictus as a secondary vector in JEV transmission in Cuddalore, Tamil Nadu lends support to the hypothesis of periodic epidemics in the region. [ABSTRACT FROM AUTHOR]