Four stereoisomers of 6-acetoxy-5-hexadecanolide, a mosquito oviposition attractant pheromone, were bioassayed for their activity on mosquitoes. Only (−)-(5R,6S) isomer was active in attractingCulex quinquefasciatus Say females for oviposition at dosages of 0.5 μg/100 ml water and above with the floating-cap method. The activity of this isomer increased 50-fold when it was applied directly to the water surface. The other three isomers, (+)-(5S,6R), (+)-(5R,6R), and (−)-(5s, 6s), were not active. The active isomer was ovipositionally attractive not only toC. quinquefasciatus but also toC. tarsalis Coquillett; however, it was 100 times more active in the former than in the latter species.Aedes aegypti (L.) andAnopheles quadrimaculatus Say were not attracted to the pheromone, thus suggesting that the pheromone is genus-specific.