We investigated the communicative function of the two notes of the advertisement call of Johnstone's whistling frog, Eleutherodactylus johnstonei, by broadcasting standard two-note calls and edited one-note calls to males in the field and to females in two-choice trials. We tested the hypothesis that each note has distinct signaling function for each sex such as has been demonstrated for the congener E. coqui. We analyzed phonotaxis in females and changes in duration and dominant frequency of each note and also in call period in the acoustic responses of males. Females were equally likely to approach a source broadcasting a standard call or the second note alone, but rejected sources broadcasting the first note alone. This indicated that the second note mediates female attraction, much as in the congener E. coqui. Male responses indicated that either note is as effective as the standard call in eliciting a change in the dominant frequency of both notes, although the modification is small quantitatively. Male behavior contrasts with that of the congener E. coqui in which the first note alone mediates male competition while the second is apparently neutral. However, we do not rule out a differential function of each note in male interactions in E. johnstonei. Other male behaviors suggestive of competition or of an imminent physical interaction (e.g., visual displays, orientation and/or approach toward the sound source) should be analyzed in further studies. Our results partially support the hypothesis of a dimorphic function of each note but are not definitive with regards to male behavior.