INTRODUCTION: The generation and selection of recombinant cell lines specifically designed to express high picomolar levels of heterologous G-protein-coupled receptors can lead to loss of ligand-dependent functional activity. As a result, the clonal selection of a suitable host model and/or lower receptor expression levels within the same cell system becomes important especially when a functional assay is necessary to evaluate the pharmacological potencies of ligands at the receptor site. To address this question, we examined the utility of various signal transducers to restore the functional capacity of a high expressing human 5-HT(6) receptor CHODUKX system. METHODS: The plasmids for human 5-HT(6) receptor and full-length human G(s), G(olf) and rat adenylyl cyclase isoforms 2 (rAC2) and 5 were obtained by PCR. The h5-HT(6) receptor pHTop plasmid was stably transfected into a CHODUKX cell line to generate an h5-HT(6) expressing clone. h5-HT(6) CHODUKX cells were transfected with signaling components and functional cAMP responses measured. rAC2 was selected to generate a double stable h5-HT(6) receptor/rAC2 pHTop CHODUKX line. RESULTS: The h5-HT(6) receptor CHODUKX line was a high receptor expressor (>2 pmol/mg protein) but an extremely poor ligand-dependent functional responder, failing to produce the appropriate cAMP signal upon addition of selective agonists. We found that stable co-expression of rAC2 with h5-HT(6) receptor in the CHODUKX cell line displayed dose-dependent cAMP accumulation following agonist treatment. The pharmacological profile of several agonists in the h5-HT(6) receptor/rAC2 cell line was consistent with an h5-HT(6)-like receptor-mediated event. DISCUSSION: We provide evidence for restoration of functional capacity in a heterologous G(s)-coupled 5-HT(6)/AC2 CHODUKX expression system. We discuss the broader value of a stable AC2-expressing CHODUKX cell line in which the generation of high expressing GPCR receptor/AC2 lines can retain their functional responsiveness and provide pharmacological drug comparisons between the same host line for screening purposes and measurement of multiple cellular parameters.