Males of the viviparous teleost fish Gambusia affinis copulate with females by using a specialized anal fin, or gonopodium. When female G. affinis were placed in a shallow transparent tank which was then floated on the surface of a larger aquarium housing male G. affinis, the males frequently attempted to copulate with females housed in the smaller quarantine tank. This copulatory behavior in male G. affinis was only observed to be elicited by visual stimuli; female G. affinis have a yellow spot and black anal spots around their urogenital opening. To investigate the function of the yellow spot of the female, we examined male copulatory behavior directed towards artificial female models having a yellow, black, gray, or white spot in the genital region. Of the differently colored females tested, males spent a significantly longer time in the vicinity of the artificial model with a yellow spot. In addition, males attempted to copulate with the yellow-spotted female model more frequently than with the models of different colors. These findings revealed that the yellow spot around the female urogenital opening of G. affinis attracts males and functions as a cue for copulation.