SummaryA two‐site ELISA has been designed for the detection of sporozoite antigen in mosquitoes. Biotin‐labelled monoclonal antibodies against sporozoites and a streptavidin‐biotin‐peroxidase complex were used to visualize the antigen.Evaluation of the sensitivity and specificity of the procedure was carried out and background levels of reactivity on the basis of negative mosquitoes were calculated. The test has been deliberately kept as simple as possible for use in the tropics and was designed usingAnopheles stephensiinfected within vitrocultivatedPlasmodium falciparumgametocytes.A minimum of about 100‐350 sporozoites could be detected in mature salivary gland infections; in addition sporozoite antigen was detected in mosquitoes several days before the entry of sporozoites into the salivary glands. No reaction was demonstrable either with bloodstage or ookinete antigens ofP. falciparum, or with mosquitoes carrying sporozoites of other plasmodial species. The number of sporozoites in positive mosquitoes and the generating capacity of a single oocyst could be assessed by the use of a calibration curve based on dilution data of a known sporozoite suspension. It was found that a single oocyst can produce about 10 000 sporozoite eq