Two parent clones of a gamma-hydroxybutyrate (GHB) receptor, C12K32 and GHBh1, were isolated from a human frontal cortex cDNA library. The two clones differ by a deleted cytosine in C12K32. CHO cells transfected with either C12K32 or GHBh1 responded positively to submicromolar GHB stimulation. However, unlike C12K32, GHBh1 desensitizes rapidly on application of low concentrations of GHB. GHB receptor properties were then studied on C12K32 expressed in CHO cells. C12K32 bound GHB with a Kd of 114 nM and has no affinity for GABA or glutamate. GHB and NCS-382 displaced [3H]GHB with an IC50 of 53 +/- 8 and 120 +/- 18 nM, respectively. In patch-clamp experiments, GHB induced a dose-dependent response with an EC50 of 130 nM. This response was antagonized by NCS-382, was not reproduced by GABA, and was sensitive to the addition of GTP-gamma-S in the recording pipette. CHO cells transfected with C12K32 exhibited GTPgamma-35S binding with an EC50 of 462 nM for GHB and an IC50 of 2.9 microM for NCS-382. The present data led to the conclusion that both C12K32 and GHBh1 are two closely related isoforms of a human GHB receptor, GHBh1, that is described in the databank as the GPCR 172A.