During the state of active sleep (AS), Clarke's column dorsal spinocerebellar tract (DSCT) neurons undergo a marked reduction in their spontaneous and excitatory amino acid (EAA)-evoked responses. The present study was performed to examine the magnitude, consistency of AS-specific suppression, and potential role of classical inhibitory amino acids GABA and glycine (GLY) in mediating this phenomenon. AS-specific suppression of DSCT neurons, expressed as the reduction in mean spontaneous firing rate during AS versus the preceding episode of wakefulness, was compared across three consecutive sleep cycles (SC), each consisting of wakefulness (W), AS, and awakening from AS (RW). Spontaneous spike rate did not differ during W or RW between SC1, SC2, and SC3. AS-specific suppression of spontaneous firing rate was found to be consistent and measured 40.3, 31.5, and 41.6% in SC1, SC2, and SC3, respectively, indicating that such inhibition is marked and stable for pharmacological analyses. Microiontophoretic experiments were performed in which the magnitude of AS-specific suppression of spontaneous spike activity was measured over three consecutive SCs: SC1-control (no drug), SC2-test (drug), and SC3-recovery (no drug). The magnitude of AS-specific suppression during SC2-test measured only 11.7 or 14.6% in the presence of GABA(A) antagonist bicuculline (BIC) or GLY antagonist strychnine (STY), respectively. Coadministration of BIC and STY abolished AS-specific suppression. AS-specific suppression of EAA-evoked DSCT spike activity was also abolished in SC2-test after BIC or STY, respectively. We conclude that GABA and GLY mediate behavioral state-specific inhibition of ascending sensory transmission via Clarke's column DSCT neurons.