Methods to discriminate echolocation calls of male and female big brown bats (Eptesicus fuscus) during the nonmating season were investigated. A total of 4018 calls from 23 bats (12 males and 11 females) were analyzed. The bat calls were recorded in natural settings in Georgia (13 bats) and Ohio (10 bats). Both hand-held and flying calls were analyzed. Calls were further divided into multiple classes based on duration. A discriminant function analysis (DFA) detected sexual differences between the calls in some situations. In particular, when calls of similar durations were compared, the results indicated that short calls may be especially useful in differentiating the sexes