(Uploaded by Plazi for the Bat Literature Project) The purpose of this study was to compare the echolocation calls of the same four individual Myotis lucifugus and Myotis leibii flying inside a closed room and when released outside. Echolocation calls were recorded using a Pettersson D980 bat detector, the high frequency output fed into a personal computer via an F2000 Control Filter and an Ines High speed card. Recorded as .wav files, recordings were analyzed with BatSoundPro. We measured call duration (DUR in ms), frequency with maximum energy (FMAX in kHz), highest frequency (HF in kHz), lowest frequency (LF in kHz), and inter-pulse interval (IPI in ms). Multivariate Analyses of Variance (MANOVA) indicated significant differences in call features between species, between settings, between species in each setting, and finally between settings for each individual. Discriminant Function Analyses (DFA) revealed that inside DUR was the most important parameter distinguishing M. lucifugus from M. leibii, with 66.3% correct classification, while outside, the two species were distinguished 78.8% of the time by LF. The data demonstrate that the same individuals flying in confined spaces change the details of their echolocation calls compared to when flying in the open. Calls produced inside are shorter in DUR and are produced at shorter IPIs than calls produced outside. FMAX differed most between the calls of M. lucifugus and M. leibii whether flying inside or outside. Differences between echolocation calls were more pronounced between setting (inside versus outside) than between species.