We compared adenylyl cyclase (AC) activation by the G protein-coupled human serotonin (5-HT) receptors 5-HT4(b) and 5-HT7(a) using an ecdysone-inducible expression system, which allowed for reproducible expression of increasing receptor densities in clonal HEK293 (EcR293) cell lines. Low constitutive expression of receptors (2-70 fmol/mg protein) was observed and could be titrated up to 50-200-fold (approximately 400-7000 fmol/mg protein) by the ecdysone analogue ponasterone A. Although 5-HT-stimulated AC activity increased with receptor density, interclonal variation precluded comparisons of coupling efficiency. Interestingly, the potency of 5-HT to stimulate AC increased with increasing receptor density only in clones expressing 5-HT4(b) receptors. The potency for 5-HT did not change in clones expressing 5-HT7(a) receptors, even though 5-HT-stimulated AC activity approached asymptotic levels. This indicates that potency of 5-HT for stimulation of AC through the 5-HT7(a) receptor is independent of receptor-Gs stoichiometry and is consistent with a model where the 5-HT7(a) receptors are tightly associated with G protein, independent of agonist binding. This supports the existence of a complex between inactive receptor and G protein, as predicted by the cubic ternary complex model. In such a system, spare receptors do not lead to increased potency of an agonist with increased receptor density.