This study was designed to functionally validate earlier described criteria for visual sleep scoring with respect to acoustical stimulus threshold for arousal. A further objective was to explore the relation between electroencephalographic (EEG) power spectrum and acoustical stimulus threshold for arousal. After habituation to an acoustical stimulus (a 1,000-Hz sine tone, increasing 1.5 dB per second for 45 seconds), values for latency to arousal after acoustical stimulus onset were analyzed. Arousal was determined based on EEG and electromyographic (EMG) criteria. There was a significant effect of sleep stage, with slow wave sleep 2 (SWS-2) having higher arousal threshold than slow wave sleep 1 (SWS-1), rapid eye movement (REM) sleep and transition type sleep. This indicates that the subdivision of nonrapid eye movement (NREM) sleep in the rat into SWS-1 and SWS-2 had functional validity in this paradigm. Time of day also had a significant effect, with lower arousal threshold in the last 2 hours (ninth and tenth hour of the light period) of the 8-hour registration period. Furthermore, there was a significant effect of EEG delta power density. Epochs with high delta power had increased arousal threshold relative to epochs with low arousal threshold. The results were consistent with the notion that delta activity is an indicator of depth within NREM sleep.