Recent studies have indicated that the glycine receptor antagonist strychnine and the gamma-aminobutyric acid type A (GABA A) receptor antagonist bicuculline reduced the rapid-eye-movement (REM) sleep-specific inhibition of sensory inflow via the dorsal spinocerebellar tract (DSCT). These findings imply that the spinal release of glycine and GABA may be due directly to the REM sleep-specific activation of reticulospinal neurons and/or glutamate-activated last-order spinal interneurons. This study used in vivo microdialysis and high-performance liquid chromatography analysis techniques to provide evidence for these possibilities. Microdialysis probes were stereotaxically positioned in the L3 spinal cord gray matter corresponding to sites where maximal cerebellar-evoked field potentials or individual DSCT and nearby spinoreticular tract (SRT) neurons could be recorded. Glutamate, glycine, and GABA levels significantly increased during REM sleep by approximately 48, 48, and 14%, respectively, compared with the control state of wakefulness. In contrast, dopamine levels significantly decreased by about 28% during REM sleep compared with wakefulness. During the state of wakefulness, electrical stimulation of the nucleus reticularis gigantocellularis (NRGc) at intensities sufficient to inhibit DSCT neuron activity, also significantly increased glutamate and glycine levels by about 69 and 45%, respectively, but not GABA or dopamine levels. We suggest that the reciprocal changes in the release of glutamate, glycine, and GABA versus dopamine during REM sleep contribute to the reduction of sensory inflow to higher brain centers via the DSCT and nearby SRT during this behavioral state. The neural pathways involved in this process likely include reticulo- and diencephalospinal and spinal interneurons.