Study Objectives: The aim of the study is to assess, in humans, transcallosal inhibition upon awakening from rapid eye movement (REM) and non-REM sleep, by paired-pulse transcranial magnetic stimulation (TMS). Design: During the daytime, a baseline session of motor evoked potentials (MEPs) was recorded. During the nighttime, the TMS sessions were administered just before sleep onset and upon awakenings from REM and stage 2 sleep, both in the early and final part of night. Setting: The sleep research laboratory at the University of Rome "La Sapienza." Participants: Ten right-handed subjects participated in the experiment for 4 consecutive sleep-recording nights. Interventions: N/A. Measurements and Results: During the daytime, a robust transcallosal inhibition was found; the MEP amplitude reduction ranged from 35% to 40%. During the nighttime, a decrease of transcallosal inhibition from right-to-left motor cortex, as compared to that from left-to-right motor cortex, was observed. The direct assessment of MEP changes, as a function of sleep stage and of the time of night, pointed to a drop of transcallosal inhibition after awakening from REM sleep. Therefore, the inhibitory activity of transcallosal fibers observed after non-REM awakening almost disappeared after REM sleep awakenings. Conclusions: The drastic drop of transcallosal inhibition after awakenings from REM sleep represents the first evidence in humans of a change of interhemispheric connectivity mediated by the corpus callosum during this sleep stage and may open new avenues for a better understanding of some aspects of sleep mechanisms (ie, dreaming function and dream mentation). Abbreviations: REM, rapid eye movement; MEP, motor evoked potentials; TMS, transcranial magnetic stimulation; EEG, electroencephalogram; ADM muscle, Abductor Digiti Minimi muscle; ISI, interstimulus intervals; MT, motor threshold; IHPP paradigm, interhemispheric paired pulse paradigm; ANOVA, analysis of variance.