Human 5-hydroxytryptamine(7) (5-HT(7)) receptors display characteristics shared with receptors believed to form a tight physical coupling with G protein in the absence of ligand. Some receptors apparently preassociated with G(i/o) and G(q/11) are reported to inhibit the signaling of other similarly coupled G protein-coupled receptors by limiting their access to activate a common G protein pool. Therefore, we determined whether 5-HT(7) receptor expression was sufficient to limit signaling of endogenously expressed G(s)-coupled receptors in human embryonic kidney (HEK) 293 cells. Using the ecdysone-inducible expression system, which allows for the titration of increasing receptor density in the same clonal cell line, we compared the effects of 5-HT(4(b)) and 5-HT(7(a,b,d)) receptor expression on adenylyl cyclase (AC) stimulation by the endogenous G(s)-coupled beta-adrenergic (betaAR) and prostanoid EP (EPR) receptors. betaAR- and EPR-stimulated AC activity was attenuated by 5-HT(7) receptor expression in both membrane preparations and intact HEK293 cells. betaAR- and EPR-stimulated AC activity was unaffected by expression of the G(s)-coupled 5-HT(4) receptor. The mechanism of this heterologous desensitization seems independent of protein kinase A activation, nor does it occur at the level of G protein activation because 1) betaAR- and EPR-stimulated AC activity was not restored to control values when Galpha(s) was overexpressed; and 2) beta(1)AR and beta(2)AR activation of Galpha(s) was unaffected by the expression of 5-HT(7) receptors. In addition, overexpression of AC isoforms was unable to rescue betaAR- and EPR-stimulated AC activity. Therefore, 5-HT(7) receptors probably limit access and/or impede activation of AC by betaAR and EP receptors. Although the 5-HT(7) receptor may preassociate with G protein and/or AC, the mechanism of this heterologous desensitization remains elusive.