Previous work has shown that stimulation of alpha 2-adrenergic receptors depresses the startle responses in rats. The present study suggests that this depressant effect involves supraspinal rather than spinal alpha 2-adrenergic receptors because intraventricular but not intrathecal infusion of the hydrophilic alpha 2-adrenergic agonist ST-91 depressed the acoustic startle reflex. To determine the point in the acoustic startle pathway where alpha 2-adrenergic receptor activation might ultimately alter neural transmission, startle responses were elicited electrically from different points along the acoustic startle pathway after systemic administration of clonidine. Clonidine depressed acoustically-elicited startle and startle elicited by electrical stimulation of the ventral cochlear nucleus to a comparable magnitude and over a similar time course. It also partially depressed startle elicited by electrical stimulation of the nucleus reticularis pontis caudalis (RPC). Taken together, these data suggest that alpha 2-adrenergic stimulation depresses startle by acting on supraspinal receptors, but that this effect is ultimately expressed, at least in part, by actions at both spinal and brainstem levels of the acoustic startle response pathway. The results are compared to other drugs known to affect the startle reflex.