Background Postural orthostatic tachycardia syndrome (POTS) is characterized by excessive heart rate increase on standing and orthostatic intolerance. Previous data indicate autoimmune involvement. We studied serum activity against G protein-coupled receptors in relation to symptoms in patients with POTS and controls using a commercial cell-based assay. Methods and Results Forty-eight patients with POTS (aged 28.6±10.5 years; 44 women) and 25 healthy individuals (aged 30.7±8.6 years; 21 women) were included. The 10-item Orthostatic Hypotension Questionnaire (OHQ) was completed by 33 patients with POTS and all controls. Human embryonic kidney 293 cells overexpressing one G protein-coupled receptor: adrenergic α receptor, adrenergic β receptor, cholinergic muscarinic type 2 receptor, and opioid receptor-like 1 were treated with sera from all patients. Receptor response was analyzed using a β-arrestin-linked transcription factor driving transgenic β-lactamase transcription by fluorescence resonance energy transfer method. Receiver operating characteristic curves were constructed. G protein-coupled receptor activation was related to OHQ indices in linear regression models. Sera from patients with POTS activated all 4 receptors to a higher degree compared with controls (<0.01 for all). The area under the curve was 0.88 (0.80-0.97, <0.001) combining all 4 receptors. Adrenergic α receptor activation associated with OHQ composite score (β=0.77 OHQ points per SD of activity, =0.009) and with reduced tolerability for prolonged standing (=0.037) and walking for short (=0.042) or long (=0.001) periods. All 4 receptors were associated with vision problems (<0.05 for all). Conclusions Our results indicate the presence of circulating proteins activating adrenergic, muscarinic, and nociceptin receptors in patients with POTS. Serum-mediated activation of these receptors has high predictive value for POTS. Activation of adrenergic α receptor is associated with orthostatic symptoms severity in patients with POTS.