G protein-coupled receptors (GPCRs) represent a class of important therapeutic targets for drug discovery. The integration of GPCRs into contemporary high-throughput functional assays is critically dependent on the presence of appropriate G proteins. Given that different GPCRs can discriminate against distinct G proteins, a universal G protein adapter is extremely desirable. In this report, the authors evaluated two highly promiscuous Galpha(16/z) chimeras, 16z25 and 16z44, for their ability to translate GPCR activation into Ca(2+) mobilization using the fluorescence imaging plate reader (FLIPR) and aequorin. A panel of 24 G(s)- or G(i)-coupled receptors was examined for their functional association with the Galpha(16/z) chimeras. Although most of the GPCRs tested were incapable of inducing Ca(2+) mobilization upon their activation by specific agonists, the introduction of 16z25 or 16z44 allowed all of these GPCRs to mediate agonist-induced Ca(2+) mobilization. In contrast, only 16 of the GPCRs tested were capable of using Galpha(16) to mobilize intracellular Ca(2+). Analysis of dose-response curves obtained with the delta-opioid, dopamine D(1), and Xenopus melatonin Mel1c receptors revealed that the Galpha(16/z) chimeras possess better sensitivity than Galpha(16) in both the FLIPR and aequorin assays. Collectively, these studies help to validate the promiscuity of the Galpha(16/z) chimeras as well as their application in contemporary drug-screening assays that are based on ligand-induced Ca(2+) mobilization.