It is currently accepted that occupancy of opioid receptors by agonists, but not antagonists, promotes the association of the receptors to guanine nucleotide binding proteins (G-proteins) and stimulates a high affinity GTPase as part of the mechanism that links the receptor-ligand complex to adenylate cyclase inhibition. In this work we report that in rat brain membranes selective delta-opioid antagonists, the peptides N,N-Diallyl-Tyr-D-Leu-Gly-Tyr-Leu-OH (Diallyl-G) and N-N-Diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI174,864), inhibit the low Km GTPase activity in a concentration dependent way. On the other hand the delta-opioid agonists D-Ala2-D-Leu5-enkephalin (DADLE) and D-Ser2-Leu5-Thr6-enkephalin stimulate dose-dependently the low Km GTPase activity in rat brain membranes. This stimulation was blocked in the presence of Diallyl-G, and reciprocally the inhibition induced by Diallyl-G was reversed by DADLE. The inhibitory effect of Diallyl-G as well as the stimulation induced by DADLE were abolished when membranes were exposed to low concentrations of N-ethylmaleimide or by ADP ribosylation with pertussis toxin which interferes with the ability of the receptor to couple to G-proteins. These observations indicate that the inhibitory effect of Diallyl-G on GTPase requires a functional G-protein and suggest that certain delta-opioid antagonists exhibit negative intrinsic activity and may have the ability to inhibit the receptor-mediated activation of G-proteins.