The presence of a previously unrecognized sporulation sequence during the early development of the microsporidium Edhazardia aedis is described from the mosquito, Aedes aegypti. This new information, together with the previous data on this species, has established that the developmental cycle of E. aedis is characterized by four sporulation sequences, two in the parental host and two in the filial generation. This is the first report of a microsporidium exhibiting four sporulation sequences. The events of gametogony, plasmogamy, nuclear association, and merogony occur almost exclusively in the gastric caeca following the initial infection and culminate with the formation of a first binucleate spore. This spore is morphologically, ultrastructurally, temporally, and functionally different from the second binucleate (transovarial) spore. Our findings suggest that the first binucleate spore is responsible for autoinfection and that its descendants lead to the formation of the second binucleate spore. (C) 1997 Academic Press.