To determine the potency and efficacy of 5-HT1F receptor ligands, a [35S]GTPgammaS binding assay was developed and optimized for the human 5-HT1F receptor. Compounds which are known to be effective in the abortive treatment of migraine were tested for efficacy and potency in this assay. Naratriptan, sumatriptan, zolmitriptan, and rizatriptan all had agonist activity. The 5-HT1F receptor ligand LY334370 (4-fluoro-N-[3-(1-methyl-4-piperidinyl)-1H-indol-5-yl]-benzamide) was the most potent compound tested with an EC50 of 2.13 +/- 0.15 nM. LY302148 (5-fluoro-3-[1-[2-(1-methyl-1H-pyrazol-4-yl)ethyl]-4-piperidinyl]-1H-ind ole), methysergide, LY306258 (3-dimethylamino-2,3,4,9-tetrahydro-1H-carbazol-6-ol), dihydroergotamine (DHE), L-694,247 and CP-122,288 were also investigated for potency and efficacy. There was a statistically significant correlation between the pEC50 for the stimulation of [35S]GTPgammaS binding and the pID50 for the inhibition of trigeminal nerve-stimulated dural plasma protein extravasation in the guinea pig. In the course of these studies, it was found that the purportedly selective 5-HT1D receptor antagonist GR127935 inhibited 5-HT1F receptor-stimulated [35S]GTPgammaS binding with a Ki of 39.6 +/- 9.5 nM. These studies demonstrate that 5-HT1F receptor-mediated stimulation of [35S]GTPgammaS binding in a clonal cell system is a reproducible, high throughput assay that is predictive of an in vivo model of 5-HT1F receptor activation.