We recently reported (Verzillo, et al. J. Neurochem: 130, 790-796, 2014) that chronic systemic morphine selectively upregulates mRNA encoding two C-terminal μ-opioid receptor (MOR) splice variants, MOR-1C1 and MOR-1B2 (MOR-1B2/-1C1). Given the known disconnects between changes in levels of mRNA and corresponding protein, it is essential to directly demonstrate that chronic opioid treatment elevates functional MOR-1B2/-1C1 protein prior to inferring relevance of these MOR variants to spinal opioid tolerance mechanisms. Accordingly, we investigated the ability of chronic opioid exposure to upregulate MOR protein in Chinese Hamster Ovary cells (CHO) stably transfected with rat MOR variants MOR-1B2, MOR-1C1, or MOR-1 (considered to be the predominant MOR). Findings revealed that chronic treatment with the clinically relevant opioids morphine, oxycodone and hydrocodone substantially upregulated membrane MOR-1B2/-1C1 protein. This upregulation was abolished by the protein synthesis inhibitor cycloheximide, eliminating contributions from receptor redistribution. The increment in MOR-1B2/-1C1 protein was paralleled by a significant increment in opioid agonist-stimulated GTPγS binding (reflective of increased aggregate MOR G protein coupling) indicating that upregulated MOR-1B2/-1C1 represented functional receptors. Strikingly, these tolerance-associated adaptations of MOR-1B2/-1C1 differed considerably from those of MOR-1. Antithetical regulation of MOR-1B2/-1C1 and MOR-1 by chronic opioids has significant implications for the design of new therapeutic agents to counteract opioid analgesic tolerance and accompanying addiction. Since chronic opioids induce MOR-1B2/-1C1 upregulation in spinal cord of males, but not females, elucidating cellular compartments and intracellular pathways mediating MOR-1B2/-1C1 upregulation and defining their unique signaling attributes would enable a precision medicinal approach to pain management and addiction therapy. This article is protected by copyright. All rights reserved.