Strict clearly mechanisms of reproductive isolation is important especially for closely related species that inhabit the same habitat. If they misidentify reproductive signals, they incur negative effects of reproductive interference between them. Thus, there should be some mechanisms to avoid reproductive interference between related species that are syntopic and simultaneity breeds in nature. Pelophylax porosus brevipodus and P. nigromaculatus are closely related species of frogs, which syntopically occur and simultanously breed in some areas of Japan. Female vocalization was recently found in these frogs. Generally, male frogs produce calls, whereas female frogs do not positively emit calls in the reproductive season. Based on these facts, I proposed a hypothesis that female calls of P. p. brevipodus and P. nigromaculatus are used for reproductive isolation. In the present study, to clarify the function of the female calls, I observed their interspecific and intraspecific male-female interactions, in particular, male responses to female calls, in the breeding season. In the first experiment, I placed one male and one female together in a test tank and observed their behavior. In the second experiment, I placed one male in the test tank and played back conspecific or heterospecific female calls. I found that the female calls of both species had no effects on male P. p. brevipodus, whereas male P. nigromaculatus became motionless in response to those calls. This result suggests that the female calls of P. p. brevipodus and P. nigromaculatus have negative effects on male P. nigromaculatus activity in the breeding season. It is likely that female calls of both species have the function as a reject signal against courting males. Especially, this function would be effective to reduce reproductive interference between female P. p. brevipodus and male P. nigromaculatus.