A direct enzyme-linked immunosorbent assay (ELISA) using species-specific monoclonal antibodies was developed for the detection and identification of Leishmania in sandflies. A titration of mock-infected Phlebotomus papatasi showed that fewer than 2000 L. major promastigotes could be detected. The percentage of infected P. papatasi collected in the field, as determined by dissection, was compared to that revealed by the ELISA. Both methods gave similar results, irrespective of whether the flies were caught by sticky papers or light-traps. The percentage of infected flies determined by either method was also similar in experimentally infected colony reared sandflies. The ELISA can be carried out using multiple species-specific antibodies, and is as accurate as identification of infected sandflies by microscopical examination. The technique should be useful for identifying sandfly species involved in transmitting different species of Leishmania, and for rapid assessment of leishmanial infection rates in endemic regions.