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The biochemical basis for the unexpected agonist-induced up-regulation of the number of radioligand binding sites for two mutated alpha1B-adrenergic receptors reported previously was investigated. Up-regulation was independent of the expression vector used and was not prevented by cycloheximide or actinomycin D, eliminating several potential transcriptional mechanisms and new receptor protein synthesis. Antagonists were also able to induce up-regulation, suggesting that ligand occupancy without signal generation was sufficient to induce the increase in binding sites. Accordingly, we hypothesized that up-regulation results from ligand-induced protection from inherent instability of these mutated receptors. Studies with receptors in isolated membranes revealed that the two mutated receptors that exhibited up-regulation in intact cells also exhibited an inherent instability of their ligand binding capacity, and binding of either agonists or antagonists to these receptors could protect against the loss of binding. In contrast, the wild-type receptor and other mutated receptors that did not exhibit up-regulation in intact cells did not exhibit instability or ligand-induced protection in isolated membranes. The occurrence of instability and protection in isolated membranes for only those mutated receptors and ligands that exhibit up-regulation in intact cells provides compelling evidence that the apparent up-regulation of binding sites in intact cells results from ligand-induced protection from an inherent instability of these G protein coupling-defective receptors. Inclusion of protease inhibitors markedly reduced the loss of binding in isolated membranes, implicating membrane-localized proteolysis as the likely mechanism for the instability.