Capsaicin, the principal pain-producing chemical in spicy peppers, binds TRPV1 to open an ion conduction pore. The four identical subunits enable TRPV1 to relay graded noxious stimuli through differential occupancy of ligand binding sites. We controlled stoichiometry and configuration of capsaicin binding by constructing tandem tetramers built of mixed wild-type (WT) or capsaicin-insensitive (S512F) subunits. By calcium imaging analysis, the tandem tetramer with all WT repeats is indistinguishable from WT TRPV1. One WT repeat is sufficient to confer capsaicin responsiveness to tandem tetramers, though with compromised capsaicin activation; binding of the first capsaicin molecule to a tandem tetramer with exactly one WT repeat was slow. The binding site closer to either the N- or the C- terminus of a single polypeptide tetramer possesses higher efficiency in gating the channel. Inclusion of a second WT repeat, even at an unfavorable position, substantially increases capsaicin potency. The potency to capsaicin of tandem tetrameric channels with three WT repeats increases further. Triple-WT tetramers, except of slightly less sensitivity than WT receptors at very low capsaicin concentration, approximate channels assembled from monomeric WT subunits. Cooperative binding exists among multiple capsaicin-binding sites, with much less efficient binding of the first than the following ligands to a TRPV1 channel. TRPV1 thus exhibits ultimate sensitivity and ability to code the entire dynamic range of agonist stimuli for maximal sensory transduction, thanks to full exploitation of all ligand-binding sites as well as a tunable gain to translate extracellular capsaicin concentrations into differential cellular responses.