The human 5-hydroxytryptamine-2C (5-HT2C) receptor has been the target of potential anxiolytics and antiobesity drugs, and its positive allosteric modulator was discovered to be l-threo-alpha-d-galacto-octopyranoside, methyl-7-chloro-6,7,8-trideoxy-6-[[(4-undecyl-2-piperidinyl)carbonyl]amino]-1-thiomonohydrochloride (2S-cis) (PNU-69176E). The drug at low micromolar concentrations (<25 microM) markedly enhanced [3H]5-HT binding (more than 300%) by increasing its affinity for low-affinity sites but with no appreciable effect on antagonist ([3H]mesulergine) binding. Functionally, PNU-69176E alone rendered receptors constitutively active, producing the pheno-types of 5-HT-activated receptors, as measured with mesulergine-sensitive guanosine 5'-O-(3-[35S]thio)triphosphate binding, transient inositol 1,4,5-triphosphate release, and [3H]inositol phosphate accumulation. These actions of PNU-69176E were observed with the human 5-HT2C receptor expressed in several mammalian cell lines (human embryonic kidney 293, NIH3T3, and SH-EP) at variable receptor densities (6 to 45 pmol/mg of protein), but not with analogous 5-HT and dopamine receptors (human 5-HT2A, 5-HT2B, 5-HT6, 5-HT7, and dopamine D2-long and D3 receptors). Structurally, PNU-69176E consists of a long alkyl chain and a polar moiety, including the alpha-d-galactopyranoside. Its analogs with shorter alkyl chains (methyl to n-hexyl instead of n-undecyl group) failed to enhance [3H]5-HT binding, and also long alkyl amides are without allosteric modulation. We propose that PNU-69176E may represent a new class of membrane receptor modulators, which probably need a long alkyl chain as a membrane anchor and target a selective polar head group to receptor modulatory sites near the membrane surface.