Distribution of Japanese encephalitis (JE) virus antigen in different tissues of mosquitoes was studied in three species viz., Culex tritaeniorhynchus, C. Vishnui and C. pseudovishnui. Overall per cent positivity was higher in the intrathoracically infected as compared to the orally infected mosquitoes, suggesting the existence of a midgut barrier. The cells at the junction of the foregut-midgut, and midgut-hindgut showed intense fluorescence from the second day post feeding onwards. This suggests that the dissemination of virus takes place from these regions of the gut. A small number of salivary glands were found negative even though fluorescence was seen in the respective head squashes, suggesting involvement of the salivary gland barrier in these mosquitoes. Though there was no difference in the salivary gland positivity between these three species, the salivary gland area positivity was high in C. tritaeniorhynchus mosquitoes. Presence of virus antigen in the ovaries and developing eggs of these three species on the third day suggests the possibility of transovarial transmission of virus even in the first gonotrophic cycle which may have epidemiological importance.