The aim of this study was to analyse whether substitution of phenylalanine in postion 124 of the human (h) 5-HT1Breceptor by cysteine, a naturally occurring variant of this receptor, modifies not only ligand binding, but also G-protein coupling and second messenger formation. Stably transfected rat C6 glioma cells, which express either the h5-HT1Bvariant receptor (VR) or the wild-type receptor (WTR) were used. In saturation experiments with [3H]5-carboxamidotryptamine ([3H]5-CT), the maximum binding (Bmax) of the VR amounted to only 60% of that to WTR. In competition experiments with 1 nm [3H]5-CT, the following 5-HT receptor ligands exhibited a higher affinity for the mutant receptor than for the WTR: L-694,247, 5-CT, 5-HT, sumatriptan (agonists listed at decreasing order of potency) and SB-224289 (a selective h5-HT1Breceptor inverse agonist with competitive antagonistic properties). In contrast, the mixed 5-HT1B/1Dreceptor antagonist GR-127935 exhibited equal affinity for both isoforms. The efficacy of L-694,247, 5-CT, 5-HT and sumatriptan in stimulating [35S]GTPγS binding (a measure of G protein coupling) to membranes of cells expressing the VR was approximately 50–65% lower compared to membranes of cells expressing the WTR, but their potency was 2.8–3.6-fold higher. SB-224289, which decreased [35S]GTPγS binding when given alone, but not GR-127935, was more potent in antagonizing the stimulatory effect of 5-CT on [35S]GTPγS binding to membranes expressing the VR compared to membranes expressing the WTR. In whole cells expressing the VR, 5-CT and sumatriptan inhibited the forskolin-stimulated cAMP accumulation 3.2-fold more potently than in cells expressing the WTR. In conclusion, our data suggest that the Phe-124-Cys mutation modifies the pharmacological properties of the h5-HT1Breceptor and may account for pharmacogenetic differences in the action of h5-HT1Breceptor ligands. Thus, the sumatriptan-induced vasospasm which occurs at low incidence as a side-effect in migraine therapy may be related to the expression of the (124-Cys)h5-HT1Breceptor in patients with additional pathogenetic factors such as coronary heart disease.