Ookinete formation from mature Plasmodium berghei gametocytes in vitro was partially inhibited by 0.05-0.1 microM atovaquone and almost totally blocked at a concentration of 0.25 microM. Microgametocyte exflagellation was not affected by atovaquone at concentrations up to 300 microM. Ookinete formation was also inhibited in culture when addition of 0.20 microM atovaquone was delayed by 4 hr, by which time DNA replication was likely to have been completed. Inhibition of ookinete formation by atovaquone was not reversed by orotic acid. Plasmodium berghei-infected Anopheles stephensi mosquitoes were fed a second blood meal 4, 7, 14, and 20 days postinfection (p.i.) from mice that had been treated with atovaquone or control diluent 8 hr previously. Atovaquone blood feeds on day 4 reduced oocyst numbers on days 6-12, although sporozoite numbers in the thorax and abdomen on day 20 were not significantly reduced. Blood feeds on day 7 slowed oocyst growth, blood feeds on day 14 did not significantly reduce sporozoite numbers, and feeds to mosquitoes on day 20 p.i. had no effect on transmission to naive mice. Sporozoite invasion of human hepatoma cells was unaffected by the presence of atovaquone.