Female Aedes aegypti are assumed to be primarily monandrous (i.e., mate only once in their lifetime), but true estimates of mating frequency have not been determined outside the laboratory. To assess polyandry in Ae. aegypti with first-generation progeny from wild mosquitoes, stable isotope semen-labeled males ((15)N or (13)C) were allowed to mate with unlabeled females in semifield enclosures (22.5 m(3)) in a dengue-endemic area in southern Mexico. On average, 14% of females were positive for both labels, indicating that they received semen from more than one male. Our results provide evidence of a small but potentially significant rate of multiple mating within a 48-hour period and provide an approach for future open-field studies of polyandry in this species. Polyandry has implications for understanding mosquito ecology, evolution, and reproductive behavior as well as genetic strategies for mosquito control.