Afferents to the median-paramedian raphe nuclear complex, which contains the B8 serotonergic cell group, were investigated in the rat with neuroanatomical and transmitter-selective retrograde labelling techniques. Injection of sensitive retrograde tracers, cholera toxin genoid or wheat germ agglutinin conjugated horseradish peroxidase into the median raphe resulted in labelling of neurons in a large number of brain regions. Projections from 26 of these regions are supported by available orthograde tracing data; the cingulate cortex, bed nucleus of stria terminalis, medial septum and diagonal band of Broca, ventral pallidum, medial and lateral preoptic areas, lateral hypothalamus, dorsomedial nucleus of hypothalamus, lateral habenula, interpeduncular nucleus, substantia nigra, central (periaqueductal) gray, and laterodorsal tegmental nucleus seem to represent major sources of afferents to the median-paramedian raphe complex. Retrogradely labelled cells were also observed in a number of regions for which anterograde tracing data are not available, including the perifornical hypothalamic nucleus, ventral premammillary nucleus, supramammillary and submammillothalamic nuclei and the B9 area. Possible excitatory amino acid afferents were identified with retrograde D-[3H]aspartate labelling. Microinjection of D-[3H]aspartate at a low concentration, 10(-4) M in 50 nl, resulted in retrograde labelling of a limited number of median raphe afferents. The most prominent labelling was observed in the lateral habenula and the interpeduncular nucleus, but retrogradely labelled cells were also noted in the medial and lateral preoptic areas, lateral and dorsal hypothalamus, ventral tegmental area, laterodorsal tegmental nucleus, medial parabrachial nucleus, and the pontine tegmentum. After injections of 10(-3) M D-[3H]aspartate selective labelling also appeared in more distant afferent regions, including cells in cingulate cortex, and in some regions located at shorter distances, such as the supramammillary nucleus. Injections of D-[3H]aspartate at high concentration, 10(-2) M, resulted in the appearance of weakly to moderately labelled cells in most afferent areas which were devoid of labelled cells after injections of lower concentrations, suggesting that this labelling may be non-specific. It was concluded that the median-paramedian raphe receives afferents from a large number of forebrain and hypothalamic regions, while relatively few brain stem regions project to this nuclear complex. The selectivity of retrograde labelling with D-[3H]aspartate was found to be concentration dependent, and it is suggested that the connections showing high affinity for D-[3H]aspartate may use excitatory amino acids as transmitters. Excitatory amino acid inputs from lateral habenula and interpeduncular nucleus may play predominant roles in the control of ascending serotonergic and non-serotonergic projections originating in the median and paramedian raphe nuclei.