Serotonergic (5-HT) neurons of the brainstem dorsal raphe nucleus (DRN) have been implicated in a diversity of physiological and behavioral processes in vertebrates. However, despite extensive information about the intrinsic properties and the efferent projections of this neurochemical system, little information is available regarding the afferents that control its activity. This study investigated the neurotransmitters that regulate the activity of DRN-5-HT neurons under physiologically relevant conditions, by utilizing microiontophoresis in combination with single-unit recordings in the awake, head-restrained cat. This made it possible to examine the direct effects of neurotransmitters on DRN-5-HT neuronal activity, and, through the use of specific antagonists, to study the roles of these neurotransmitter inputs during physiological conditions that influence DRN-5-HT neuronal activity. The results indicate that (1) iontophoretic application of the GABA antagonist bicuculline reversed the typical suppression of neuronal activity seen during slow wave sleep, but had no effect on maintained activity during wakefulness. The suppression of neuronal activity during REM sleep was generally unaffected by application of bicuculline. This suggests a role for a GABAergic input to DRN-5-HT neurons in controlling some aspects of their state-dependent activity. (2) Iontophoretic application of the excitatory amino acid (EAA) antagonist kynurenic acid reduced the magnitude of the neuronal response evoked by phasic auditory stimuli, but had no effect on the spontaneous activity of these neurons, suggesting a role for an EAA input to the DRN in mediating the response to phasic sensory stimuli.(ABSTRACT TRUNCATED AT 250 WORDS)