External fertilization makes male anurans susceptible to direct intrasexual competition for fertilization opportunities at the egg mass. The red-eyed treefrog, Agalychnis callidryas, is one species in which pairs of males appear to simultaneously fertilize the clutch of a Single female. DNA fingerprinting revealed the presence of multiple paternity in two egg clutches examined from two matings involving a female with two males. The breeding biology of females and the potential costs and benefits of mating with multiple males were examined. Females were found to decrease the number of eggs in matings with multiple males. In addition, amplexed females moving toward Oviposition sites avoided secondary males by moving when approached by secondary males. Mortality to the eggs as a result of multiple males attempting to amplex females is suggested as the reason females avoid multiple males. Males were found to exhibit calling site defense from other males. Males used a combination of auditory and a visual behavior in defending calling sites. The call types are described and the contexts within which calls occur is discussed. Density of frogs was found to be a better indicator of the occurrence of matings involving multiple males than the operational sex ratio (number of males/number of females).