We describe the first validated scintillation proximity assay (SPA) binding method for quantitation of 3 H-labeled d-lysergic acid diethylamide (LSD) binding to recombinant human 5-hydroxytryptamine 6 (5-HT 6 ) receptors expressed in Chinese hamster ovary (CHO)–Dukx and HeLa cells. The assay was developed using intact cells as a receptor source because membrane fractions derived from these cells failed to discern specific binding from a high level of nonspecific binding. The pharmacological binding profile of seven 5-HT 6 agonists and antagonists using intact CHO–Dukx/5-HT 6 cells in the SPA format was similar to data obtained from a filtration binding assay using HeLa/5-HT 6 membranes. K i values and rank order of potencies obtained in the SPA format were consistent with published filtration data as follows: SB-271046 (K i =1.9nM)>methiothepin (K i =6.2nM)>mianserin (K i =74.3nM)>5-methoxytryptamine (5-MeOT, K i =111nM)>5-HT (K i =150nM)>ritanserin (K i =207nM)>5-carboxamidotryptamine (5-CT, K i =704nM). Additional evaluation with four antipsychotics demonstrated strong agreement with previous literature reports. A high specific binding signal and low assay variability, as determined by Z′=0.81±0.017, make the SPA format amenable to automation and higher throughput; hence, this assay can be a viable alternative to the more labor-intensive filtration and centrifugation methods.