Primary cultures of dissociated hippocampi from fetal mice examined for the presence of binding sites for [3H]diazepam. The binding assays were done with living cells still attached to the culture dish. The cells contain high affinity binding sites for [3H]diazepam, Kd = 5 nM, which are completely inhibited with 20 nM R05-4864 but only 26% with 20 nM lorazepam. The binding was inhibited by purinergic compounds and by quinidine. The living cell did not exhibit increased binding of [3H]diazepam in the presence of GABA and in fact a slight decrease in binding was found. This was also found when live, intact C6 glial cells were investigated. These observations suggest that the use of living cells to study the benzodiazepine receptor is valuable and maybe necessary to fully characterize this receptor.