1 Naloxone benzoylhydrazone (NalBzoH) has initially been developed as an agonist of the pharmacologically defined kappa3-opioid receptor and has recently been employed as an antagonist at the opioid receptor-like (ORL1) receptor. In the present study, we investigated the ability of NalBzoH to elicit agonist-like effects on receptor signalling in distinct layers of rat olfactory bulb, a brain region where we have demonstrated the presence of opioid and ORL1 receptors coupled to both stimulation and inhibition of cyclic AMP formation. 2 In membranes of the olfactory nerve-glomerular layer (ON-GL), external plexiform layer (EPL) and granule cell layer (GRL), NalBzoH elicited a concentration-dependent stimulation of guanosine-5'-O-(3-[35S]-thio)triphosphate ([35S]GTPgammaS) binding with pEC50 values ranging from 7.36 to 7.86, whereas the kappa1-opioid receptor agonists (-)-U-50,488 and U-69,593 were inactive. 3 In membranes of GRL, but not ON-GL and EPL, NalBzoH stimulated basal adenylyl cyclase activity by 40% with a pEC50 of 8.14, and significantly potentiated the net enzyme stimulation elicited by corticotropin-releasing hormone and pituitary adenylate cyclase-activating peptide 38. Pertussis toxin prevented the NalBzoH stimulations of [35S]GTPgammaS binding and adenylyl cyclase activity. 4 In membranes of EPL and GRL, but not ON-GL, NalBzoH elicited a concentration-dependent inhibition of forskolin-stimulated adenylyl cyclase activity with pEC50 values of 8.07 and 8.08, respectively. 5 At concentrations that completely blocked the actions of nociceptin/orphanin FQ (N/OFQ), the ORL1 receptor antagonists CompB and [Nphe1]N/OFQ(1-13)NH2 failed to antagonize either the stimulatory or the inhibitory effect of NalBzoH on cyclic AMP formation. Similarly, the kappa1-opioid receptor antagonist nor-binaltorphimine counteracted the NalBzoH effects with relatively low potencies (pKi values=7.67-8.09). 6 Conversely, the selective delta-opioid receptor antagonist TIPP (pKi=9.10) and the selective mu-opioid receptor antagonist CTAP (pKi=8.27) reduced the inhibitory effect of NalBzoH by 70 and 30%, respectively. Moreover, TIPP and CTAP potently inhibited the NalBzoH stimulation of cyclic AMP, each antagonist maximally causing 50% blockade of the agonist response. 7These data demonstrate that in the olfactory bulb NalBzoH activates receptor signalling by acting through delta- and mu-opioid receptors and independently of ORL1 and kappa1-opioid receptors.