Female Anopheles mosquitoes usually mate only once, but mating is seldom seen in small containers containing only one female and male. Therefore, matings are often performed among many adults in large cages or by forced copulation. Isolated-pair mating of Anopheles gambiae G3 strain–derived mosquitoes without forced copulation in small vials is described. We observed that the experimental variables eye color and male number were significant factors in the mating frequency. Females mated more frequently when three males were present over only one male. White-eyed females were more likely to be mated than wild-eyed females, but wild males mated more frequently than did white-eyed males. Experiments were also conducted to determine when mating was occurring by using wild-eye-color mosquitoes in isolated pairs. Almost no matings were observed before day 6 rather than the frequencies typically observed after 1–2 d in standard large-cage matings among large numbers of adults.