G-protein-coupled receptors are a major target for potential therapeutics; yet, a large number of these receptors couple to the G_ipathway, generating signals that are difficult to detect. We have combined chimeric G proteins, automated sample handling, and simultaneous 96-well fluorometric imaging to develop a high-throughput assay system for G_isignaling. The chimeric G proteins alter receptor coupling so that signaling can occur through G_qand result in mobilization of intracellular calcium stores. An automated signaling assay device, the fluorometric imaging plate reader (FLIPR), can simultaneously measure this response in real time in 96-well microplates, allowing two people to process more than 10,000 points per day. We used the chimeric G protein/FLIPR system to characterize signaling by the G_i-coupled human opioid receptors. We show that the μ, δ, and κ opioid receptors and the related nociceptin receptor, ORL1, each couple to Gα_qi5, Gα_qo5, and Gα₁₆(Gα_qi5and Gα_qo5refer to Gα_qproteins containing the five carboxyl-terminal amino acids from Gα_iand Gα_o, respectively) and that different receptor/G protein combinations show different levels of maximal activation. We tested 31 opioid ligands for agonist activity at the opioid receptors (124 ligand–receptor combinations); all 31 activated at least one receptor type, and several activated multiple receptors with differing potencies. This high-throughput assay could be useful for dissecting the complex ligand–receptor relationships that are common in nature.