The synthesis of 5-thienyl and 5-furyl-substituted benzodiazepines is described. These compounds were employed to probe the lipophilic pocket (L3) of the benzodiazepine receptor (BzR) and to determine the effect of occupation of L3on biological activity. Of the new analogs synthesized, the 5-(2-thienyl)-benzodiazepines 6aand 7adisplayed high affinity for the BzR (IC5028 and 18 nM, respectively) and exhibited both anticonvulsant (ED50~ 9 and 3 mg/kg) and muscle relaxant (ED50~ 10 and 7 mg /kg) activity. The 5-(3-thienyl)benzodiazepines 6d and 7d displayed only moderate affinity for the BzR (IC50140 and 110 nM) and exhibited no biological activity (no anticonvulsant or muscle relaxant activity) at doses up to 40 mg/kg. The 5-(2-furyl)benzodiazepines (6b, 7b, 19band 20b) exhibit low affinities for the BzR. These in vitroand in vivofindings are consistent with our model suggesting that pocket L3is very sensitive to lipophilic effects. Thus, decreasing the lipophilicity of functional groups which occupy this region decreases ligand affinity at BzR. The 2′-halogen (F or Cl) substituent of the 5-phenylbenzodiazepines increases ligand affinity in vitrobecause the active conformation of the phenyl N(4)=C(5)-C(1′)=C(2′) moiety is synrather than anti. The synconformation permits the 2′-halogen (F or Cl) atom to interact at the hydrogen bonding site H2and form a stable three-centered hydrogen bond in the proposed ligand binding cleft. The 3-thienyl and 2-furyl groups decrease the lipophilicity of the substituent which occupies L3but do not form a hydrogen bond at H2, thus resulting in a diminished affinity at BzR.