S-(+)-Norfenfluramine (SNF)-an active metabolite of the now-banned anorexigen fenfluramine-has been implicated in the drug's appetite-suppressing actions and its life-threatening cardiovascular side effects. SNF reduces appetite through serotonin 5-HT(2C) receptor activation; it causes cardiopulmonary side effects through 5-HT(2B) receptor activation. Thus, we attempted to identify molecular determinants of SNF binding to 5-HT(2B) receptors distinct from those underlying SNF-5-HT(2C/2A) receptor interactions. Mutagenesis implicated Val2.53 in SNF binding to 5-HT(2B) receptors. Ligand docking simulations suggested both Val2.53 gamma-methyl groups form stabilizing van der Waals' (vdW) interactions with the alpha-methyl group of SNF. A V2.53L mutation induced a 17-fold decrease in affinity; molecular dynamics (MD) simulations suggested that this decrease resulted from the loss of one 2.53-alpha-methyl group vdW interaction. Supporting this, 1) the binding of norfenfluramine (NF) analogs lacking an S-(+) alpha-methyl group (RNF and alpha-desmethyl-NF) was less sensitive to the V2.53L mutation, and 2) a V2.53A mutation decreased SNF affinity 190-fold, but decreased RNF and alpha-desmethyl-NF affinities only 16- and 45-fold, respectively. We next addressed whether the alpha-methyl group of SNF contributes to 5-HT(2C/2A) receptor affinity. Removal of the alpha-methyl group (RNF and alpha-desmethyl-NF), which reduced 5-HT(2B) receptor binding 3-fold, did not affect 5-HT(2C/2A) receptor binding. An alpha-ethyl substituent (alpha-ethyl-NF), which decreased 5-HT(2B) receptor affinity 46-fold, reduced 5-HT(2C) and 5-HT(2A) receptor binding by 14- and 5-fold, respectively. Finally, we determined that residue 2.53 affects SNF potency and efficacy at 5-HT(2B) receptors but not at 5-HT(2C) and 5-HT(2A) receptors. In conclusion, vdW interactions between residue 2.53 and the alpha-methyl group of SNF contribute to the ligand's 5-HT(2) receptor subtype-selective pharmacology.