Transient expression of apoaequorin in Chinese hamster ovary (CHO) cells and reconstitution with the co-factor coelenterazine resulted in a large, concentration-dependent agonist-mediated luminescent response following cotransfection with the endothelin ETA, angiotensin ATII, thyrotropin-releasing hormone (TRH), and neurokinin NK1 receptors, all of which interact pre-dominantly with the G alpha q-like phosphoinositidase-linked G-proteins. A substantially greater luminescence was obtained with mitochondrially targeted apoaequorin compared to cytoplasmically expressed apoaequorin. To generate a system amenable for the study of agonist activity at virtually any G-protein-coupled receptor the alpha subunit of the receptor promiscuous G-protein G alpha 16 was either transiently or stably expressed in CHO cells together with apoaequorin. In cells expressing G alpha 16, but not in its absence, agonists at a series of receptors which normally interact with either G alpha s or G alpha i were now able to cause a luminescent response from mitochondrially targeted apoaequorin. In the case of the A1 adenosine receptor, this response was clearly a result of activation of G alpha 16 and not a consequence of the release of the G alpha i-associated beta/gamma complex, as the luminescent response was unaffected by pertussis toxin treatment of the cells, whereas agonist-mediated inhibition of adenylyl cyclase activity was attenuated. These studies describe the use of coexpressed apoaequorin as a reporter for G-protein-coupled receptor-mediated calcium signaling. Furthermore, coexpression of G alpha 16 and apoaequorin provides a basis for a generic mammalian cell microplate assay for the assessment of agonist action at virtually any G-protein-coupled receptor, including orphan receptors for which the physiological signal transduction mechanism may be unknown.