AbstractAn alignment of serotonin [5‐hydroxytryptamine (5‐HT)] G protein‐coupled receptors identified a lysine at position 4.45 (helix 4) and a small polar residue (serine or cysteine) at 7.45 (helix 7) that occur exclusively in the 5‐HT2 receptor family. Other serotonin receptors have a hydrophobic amino acid, typically a methionine, at 4.45 and an invariant asparagine at 7.45. The functional significance of these class‐specific substitutions was tested by site‐directed mutagenesis of two distantly related 5‐HT2 receptors, Caenorhabditis elegans 5‐HT2ce and rat 5‐HT2C. Residues 4.45 and 7.45 were each mutated to a methionine and asparagine, respectively, or an alanine and the resulting constructs were tested for activity. A K4.45M mutation decreased serotonin‐dependent activity (Emax) of the rat 5‐HT2C receptor by 60% and that of the C. elegans homologue by 40%, as determined by a fluorometric plate‐based calcium assay. The rat mutant also exhibited nearly sixfold higher agonist binding affinity and significantly lower constitutive activity compared with wildtype. Mutagenesis of S7.45 in the C. elegans receptor increased serotonin binding affinity by up to 25‐fold and decreased Emax by up to 65%. The same mutations of the cognate C7.45 in rat 5‐HT2C produced a smaller fourfold change in the affinity for serotonin and decreased agonist efficacy by up to 50%. Substitutions of S/C7.45 did not produce a significant change in the basal activity of either receptor. All mutants tested exhibited levels of receptor expression similar to the corresponding wildtype based on measurements of specific [3H]‐mesulergine binding or flow cytometry analyses. Taken together, these results suggest that K4.45 and S/C7.45 play an important role in the conformational rearrangements leading to agonist‐induced activation of 5‐HT2 receptors.