The present investigation provides evidence for serotonergic involvement in the inhibition of the acoustic startle reaction which results from the presentation of neutral stimuli (prepulses) shortly before reflex elicitation. While the specific serotonin (5-HT) synthesis inhibitor p-Chlorophenylalanine did not affect the response, a large amine-depleting dose of reserpine enhanced the reaction elicited under stable (control) stimulus conditions (no prepulse delivered). Selective replacement of 5-HT by administration of the l-amino acid precursor and a monoamine oxidase inhibitor further enhanced control startle amplitude and also eliminated prepulse inhibition. Chemical assays indicated especially large increases in 5-HT levels in the cortex and brainstem. Administration of the 5-HT precursor in the non-reserpinized rat both increased control startle levels and also interfered with prepulse inhibition although not as completely as in the reserpinized animal. The results indicate that 5-HT has a facilitatory effect on the reflex and suggest that a catecholaminergic system is also involved in determining reflex amplitude.