Sarco(endo)plasmic reticulum calcium ATPase (SERCA)2b maintains the cellular Ca(2+) homeostasis by transferring Ca(2+) from the cytosol to the lumen of the endoplasmic reticulum (ER). Recently, SERCA2b has also been shown to be involved in the biosynthesis of secreted and membrane proteins via direct protein-protein interactions, involving components of the ER folding and quality-control machinery, as well as newly synthesized G protein-coupled receptors. Here we demonstrate that the human delta opioid receptor (hdeltaOR) exists in a ternary complex with SERCA2b and the ER molecular chaperone calnexin. The interaction between SERCA2b and hdeltaOR in vivo did not require calnexin as it was independent of the C-terminal calnexin-interacting domain of SERCA2b. However, the receptor was able to mediate co-immunoprecipitation of calnexin with the C-terminally truncated SERCA2b. The association of SERCA2b with hdeltaOR was regulated in vitro by Ca(2+) and ATP in a manner that was opposite to the calnexin-hdeltaOR interaction. Importantly, co-expression of the catalytically inactive SERCA2b(D351A) or calnexin binding-compromised SERCA2bDeltaC mutants with the receptor decreased the expression of mature receptors in a manner that did not directly relate to changes in the ER Ca(2+) concentration. We conclude that dynamic interactions among SERCA2b, calnexin and the hdeltaOR precursor orchestrate receptor biogenesis and are regulated by Ca(2+) and ATP. We further hypothesize that the primary role of SERCA2b in this process is to act as a Ca(2+) sensor in the vicinity of active translocons, integrating protein folding with local fluctuations of ER Ca(2+) levels.