The possible role that the hypothalamic arcuate nucleus might play in mediating the increase in paraventricular nucleus corticotropin-releasing hormone mRNA levels following adrenalectomy was investigated in two series of experiments. In the first series in situ hybridization histochemistry was used to quantify levels of eight accurate nucleus neuropeptide and neurotransmitter mRNAs in neurons that potentially relay adrenal steroid feedback to the paraventricular nucleus. In the second series of experiments, arcuate neuropeptidergic projections to the hypothalamic paraventricular nucleus were characterized using retrograde tracing in combination with in situ hybridization histochemistry. Despite an increase in paraventricular nucleus corticotropin-releasing hormone (60%) and pituitary proopiomelanocortin mRNA levels (sixfold), arcuate mRNA levels for proopiomelanocortin, neuropeptide Y, somatostatin, galanin, dynorphin, tyrosine hydroxylase, glutamate decarboxylase, and the glucocorticoid receptor were unchanged 14 days following adrenalectomy. Neuropeptidergic characterization of arcuatoparaventricular projections was achieved by injection of the retrograde tracer fluorogold into the paraventricular nucleus; retrogradely labeled neurons were characterized with polyclonal antisera against fluorogold in combination with oligonucleotide probes directed against neuropeptide Y, proopiomelanocortin, or somatostatin. Out of these three arcuate neuropeptide Y mRNA was contained in 18% of the fluorogold-positive neurons in the arcuate, proopiomelanocortin mRNA was contained in 8%, and somatostatin mRNA was contained in 6%. Overall, the results from both experiments suggest that the arcuatoparaventricular neuropeptide Y, proopiomelanocortin, and somatostatin projections are not sensitive to a chronic (14 day) lack of adrenal steroids. These projections as well as the other arcuate neurotransmitter and neuropeptide systems appear not to contribute to the persistent elevations in paraventricular nucleus corticotropin-releasing hormone mRNA levels or pituitary proopiomelanocortin mRNA levels found in 14 day adrenalectomized rats.