The indirect immunofluorescence technique was used to study the relation between corticotropin-releasing factor (CRF) and GABAergic neurons in the rat hypothalamic paraventricular nucleus (PVN). In colchicine-pretreated animals, glutamic acid decarboxylase (GAD)- and GABA-immunoreactive (IR) neurons were observed within the medial part of the parvocellular division of the PVN as well as surrounding the nucleus itself. In general, the GAD antiserum, as compared to the GABA antiserum, revealed stronger IR cells and a higher number of cells in the PVN. CRF-IR cells were observed throughout the whole rostrocaudal extension of the PVN, but predominantly in its dorsal medial parvocellular part. Adjacent sections stained, respectively, with antisera against CRF, GAD or GABA, revealed overlapping distributional patterns within the parvocellular part of the PVN. Employing a direct double-staining technique with sheep GAD and rabbit CRF antisera, it was demonstrated that both GAD and CRF immunoreactivities occurred in the same neurons, particularly in the dorsomedial aspect of the parvocellular PVN. GAD-positive neurons located in the ventromedial parvocellular PVN, periventricular nucleus and surrounding the PVN lacked CRF immunoreactivity. In addition, with an elution-restaining technique it was possible to demonstrate that also GABA immunoreactivity was present in some CRF neurons in the dorsomedial parvocellular PVN. Intravenous injection of the retrograde tracer Fast Blue resulted in labelling of neurons in the periventricular area and in both the parvocellular and magnocellular division of the PVN. After processing the sections for GAD/CRF immunohistochemistry, it was possible to demonstrate the presence of retrogradely labelled GAD/CRF cells, pointing at a PVN median eminence projection for these neurons. The present findings demonstrate that a subpopulation of the CRF-containing neurons in the parvocellular division of the PVN in addition are GABAergic, thereby indicating a possible corelease of these compounds, presumably in the median eminence. Possible interactions of GABA within the hypothalamo-pituitary-adrenal axis are discussed.