In the present study, we examined whether the human immunodeficiency virus type I (HIV-I) gp120 coat protein can modulate corticotropin releasing factor (CRF) secretion by using the incubation of rat hypothalamic explants as an in vitro model. Treatment of the hypothalamic fragments with recombinant gp120 resulted in a time- and concentration-dependent increase in CRF release. The maximal dose of 10 nM gp120 increased CRF release by 56.4% after 1 h, and 78.4% after 3 h, as compared with their respective controls. The intra-hypothalamic amount of CRF was also increased by 54.7% and 77.3% vs. controls after 1 and 3 h, respectively. Moreover, the action of gp120 was blocked by pretreatment with cycloheximide, suggesting that the viral protein modulates CRF secretion via an increase in its synthesis. We also investigated the effects of gp120 on CRF gene expression. RNase protection analyses of total RNA isolated from the explants indicated that 10 nM gp120 significantly increases CRF mRNA in a time-dependent manner. Furthermore, gp120 did not modify CRF mRNA stability, suggesting that the viral protein modulates CRF gene expression at the transcriptional level. Analysis of the mechanisms that mediate gp120-induced CRF synthesis was conducted. The incubation of the explants with recombinant interleukin-1 (IL-1) type I receptor antagonist (hrIL-1 ra) did not antagonize the actions of gp120 at 1 and 3 h, indicating that the effect of the latter is independent of IL-1 mediated mechanisms. The involvement of some second messenger pathways was also investigated. Specific inhibitors of cAMP-PKA, cyclo-oxygenase or heme oxygenase pathways failed to antagonize the gp120-induced increase in CRF production. By contrast, incubation with nonselective inhibitors of nitric oxide synthase (NOS), L-NAME and L-NNA, or aminoguanidine (AG), a selective inhibitor of inducible NOS (iNOS), blocked CRF release and, AG, its mRNA accumulation, stimulated by gp120, whereas selective inhibitors of endothelial and neuronal NOS had no effect. In addition, only L-NAME, L-NNA and AG were able to inhibit the gp120-stimulated production of nitrites. These results indicate that gp120 directly stimulates CRF gene expression and peptide synthesis from the rat hypothalamus in vitro via the activation of iNOS. Therefore, the actions of this viral protein on the HPA axis may, in part, reflect its ability to modulate CRF synthesis.