The Ca(2+) modulation in the dorsal raphe nucleus (DRN) plays an important role in sleep-wake regulation. Calmodulin-dependent kinase II (CaMKII) is an important signal-transducing molecule that is activated by Ca(2+) . This study investigated the effects of intracellular Ca(2+) /CaMKII signaling in the DRN on sleep-wake states in rats. Maximum and minimum CaMKII phosphorylation was detected at Zeitgeber time 21 (ZT 21; wakefulness state) and ZT 3 (sleep state), respectively, across the light-dark rhythm in the DRN in rats. Six-hour sleep deprivation significantly reduced CaMKII phosphorylation in the DRN. Microinjection of the CAMKII activation inhibitor KN-93 (5 or 10 nmol) into the DRN suppressed wakefulness and enhanced rapid-eye-movement sleep (REMS) and non-REM sleep (NREMS). Application of a high dose of KN-93 (10 nmol) increased slow-wave sleep (SWS) time, SWS bouts, the mean duration of SWS, the percentage of SWS relative to total sleep, and delta power density during NREMS. Microinjection of CaCl2 (50 nmol) in the DRN increased CaMKII phosphorylation and decreased NREMS, SWS, and REMS. KN-93 abolished the inhibitory effects of CaCl2 on NREMS, SWS, and REMS. These data indicate a novel wake-promoting and sleep-suppressing role for the Ca(2+) /CaMKII signaling pathway in DRN neurons. We propose that the intracellular Ca(2+) /CaMKII signaling in the dorsal raphe nucleus (DRN) plays wake-promoting and sleep-suppressing role in rats. Intra-DRN application of KN-93 (CaMKII activation inhibitor) suppressed wakefulness and enhanced rapid-eye-movement sleep (REMS) and non-REMS (NREMS). Intra-DRN application of CaCl2 attenuated REMS and NREMS. We think these findings should provide a novel cellular and molecular mechanism of sleep-wake regulation.