The compound N-piperidinyl-[8-chloro-1-(2,4-dichlorophenyl)-1,4,5,6-tetrahydrobenzo [6,7]cyclohepta[1,2-c]pyrazole-3-carboxamide] (NESS 0327) was synthesized and evaluated for binding affinity toward cannabinoid CB1 and CB2 receptor. NESS 0327 exhibited a stronger selectivity for CB1 receptor compared with N-piperidinyl-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (SR 141716A), showing a much higher affinity for CB1 receptor (Ki = 350 +/- 5 fM and 1.8 +/- 0.075 nM, respectively) and a higher affinity for the CB2 receptor (Ki = 21 +/- 0.5 nM and 514 +/- 30 nM, respectively). Affinity ratios demonstrated that NESS 0327 was more than 60,000-fold selective for the CB1 receptor, whereas SR 141716A only 285-fold. NESS 0327 alone did not produce concentration-dependent stimulation of guanosine 5'-O-(3-[35S]thio)-triphosphate ([35S]GTPgammaS) binding in rat cerebella membranes. Conversely, NESS 0327 antagonized [R(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl)methyl]pyrolol [1,2,3-de]-1,4-benzoxazin-yl]-(1-naphthalenyl)methanone mesylate] (WIN 55,212-2)-stimulated [35S]GTPgammaS binding. In functional assay, NESS 0327 antagonized the inhibitory effects of WIN 55,212-2 on electrically evoked contractions in mouse isolated vas deferens preparations with pA2 value of 12.46 +/- 0.23. In vivo studies indicated that NESS 0327 antagonized the antinociceptive effect produced by WIN 55,212-2 (2 mg/kg s.c.) in both tail-flick (ID50 = 0.042 +/- 0.01 mg/kg i.p.) and hot-plate test (ID50 = 0.018 +/- 0.006 mg/kg i.p.). These results indicated that NESS 0327 is a novel cannabinoid antagonist with high selectivity for the cannabinoid CB1 receptor.