We have recently shown that CCKB receptor antagonists such as PD-134,308, 4-([2-[[3-(1H-indol-3-yl)-2-methyl-1-oxo-2-[[tricyclo[3.3.1.1]dec - 2-yloxy)carbonyl]amino]propyl]amino]-1-phenylethyl]amino)-4-oxo[R- (R*,R*)]-butanoate-N-methyl-D-glucamine, are able to strongly potentiate antinociception induced by endogenous enkephalins, protected from degrading enzymes by the mixed inhibitor RB 101, N-[(R,S)-2-benzyl-3[(S)-(2-amino-4- methylthio)butyldithio]-1-oxopropyl)-L-phenylalanine benzyl ester, at both spinal and supraspinal levels. In this study, the duration of this facilitatory response and the possible development of tolerance to this synergistic effect were investigated in the rat tail-flick test after acute and chronic treatment with PD-134,308 and RB 101. PD-134,308 facilitated and prolonged the antinociceptive responses induced by RB 101 (20 mg/kg, i.v.). The duration of the effect induced by PD-134,308 was also investigated by injecting this compound at different times before RB 101 administration. In the case of the tail-flick test, the improvement of RB 101 antinociceptive response was still significant 6 h after PD-134,308 (3 mg/kg, i.p.), whereas in the hot-plate test, this enhancement was only effective for 3 h after CCKB receptor antagonist administration. In the case of a repeated administration of RB 101, the potentiation induced by PD-134,308 on the antinociceptive effect produced by the first injection of RB 101 (20 mg/kg, i.v.), was found almost identical after a second administration of RB 101 performed 190 min later. Chronic administration of RB 101 (20 mg/kg, i.v.) plus PD-134,308 (3 mg/kg, i.p.) administered for 5 days both once or twice per day, did not induce the development of tolerance to antinociception at the peak effect time. However, a decrease in the duration of the antinociceptive response was observed. These results indicate that the potent and long-lasting antinociceptive response induced by the coadministration of the peptidase inhibitor and the CCKB receptor antagonist could have interesting perspectives in the clinical treatment of pain.