In this study we investigated the effects of possible modulatory transmitters on acoustically responsive neurons of the caudal pontine reticular nucleus (PnC). From previous work in our laboratory it has been suggested that the acoustically responsive giant neurons of this nucleus are the sensorimotor interface mediating the acoustic startle response. Furthermore they are the site of some of the modulatory influence impinging on this response. Besides a possibly glutamatergic excitation from the amygdala a cholinergic input from the midbrain has been described which may use substance P as cotransmitter. Therefore we used electrophysiological and histochemical methods to study this possible modulatory influence in the caudal pontine reticular nucleus. In the first part of this study we recorded extracellularly from single units in the PnC in vivo and studied the effects of iontophoretically applied transmitters. Substance P elicited a long lasting excitation. This excitatory effect of SP was potentiated by acetyl-beta-methylcholine (AMCh, an acetylcholine agonist), whereas single application of AMCh showed no uniform response. Glutamate elicited a potent brief excitation, while application of GABA showed a potent brief inhibition of PnC neurons. In the second part of this study we employed immunoperoxidase staining for substance P, which revealed a fairly dense network of substance P-immunoreactive (SP-ir) fibers in the lateral and ventral aspects of the PnC. Combining retrograde tracing and immunocytochemistry for substance P, we demonstrated that the SP-ir axons in the PnC originate mainly in the laterodorsal tegmental nucleus. We therefore conclude that activation of the laterodorsal tegmental nucleus may facilitate the acoustic startle response by a long lasting excitation of neurons in the caudal pontine reticular nucleus.