The acoustic startle reflex (ASR) in rats exhibits robust circadian modulation, with ASR amplitudes greater during subjective night. To identify the location of this modulation, startle reactions were evoked either acoustically or electrically via electrodes implanted in the primary ASR circuit. Startle amplitudes were compared at different times in the circadian cycle. In constant environmental conditions, startle amplitudes were greater in subjective night for acoustically evoked and for electrically evoked reactions from the ventral lateral lemniscus and medial longitudinal fasciculus. The results show that at least 1 site of circadian modulation must occur at some point in the circuit after the last brainstem synapse in the caudal pontine reticular formation, at the level of spinal interneurons or motoneurons or at the neuromuscular junction.