Specific endogenous [3H]flunitrazepam binding sites were identified and characterized in membranes from the human embryonic kidney (HEK) cell line 293. A large part of these binding sites exhibited an intermediate affinity for [3H]flunitrazepam and a microM affinity for diazepam, clonazepam, 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxam ide (PK 11195) or 4'-chlorodiazepam (Ro 5-4864). These sites, thus, resembled neither gamma-aminobutyric acidA (GABAA) receptor associated nor 'peripheral' benzodiazepine binding sites. A small part of the binding sites labeled by [3H]flunitrazepam seemed to belong to 'peripheral' benzodiazepine binding sites exhibiting a nM affinity for PK 11195, and another small part of the binding sites seemed to exhibit a high affinity for flunitrazepam and PK 11195. Although small amounts of mRNA for alpha 1-, beta 3- and gamma 2-subunits of GABAA receptors could be identified in HEK 293 cells, neither the actual expression of GABAA receptors in these cells nor a coassembly of endogenous subunits with transfected GABAA receptor subunits could be demonstrated by binding studies.