Class B G protein-coupled receptors (GPCRs) are peptide hormone receptors, many of which, such as parathyroid hormone receptor 1, calcitonin receptor (CTR), and corticotropin-releasing factor receptor (CRF1R), are established or emerging therapeutic targets. Previously, we showed that extracellular ATP and related molecules act as positive modulators of parathyroid hormone receptor 1 signaling through an undefined mechanism. Here, we investigated whether ATP enhances signaling by other members of the class B family of GPCRs. Cyclic AMP (cAMP) accumulation was monitored in cells expressing a bioluminescent sensor. Extracellular ATP, which did not induce cAMP accumulation on its own, potentiated agonist-induced cAMP accumulation mediated by CTR, CRF1R, calcitonin receptor-like receptor, pituitary adenylyl cyclase-activating polypeptide receptor 1, and vasoactive intestinal peptide receptors 1 and 2. ATP induced a comparable effect on agonist-stimulated recruitment of β-arrestin to pituitary adenylyl cyclase-activating polypeptide receptor 1. Depending on the receptor and agonist, ATP increased agonist potency by up to 50-fold. The enhancing effect of ATP was mimicked by cytidine 5'-monophosphate, ruling out involvement of purinergic receptors, ATPase activity, or ectokinase activity. For certain receptors (CTR, calcitonin receptor-like receptor + receptor activity-modifying protein 1, and CRF1R), there were temporal lags of up to 30 minutes following agonist application before maximal rates of cAMP accumulation were reached. Lag duration decreased with increasing agonist concentration, suggesting an inverse relationship with receptor occupancy. ATP virtually abolished this temporal lag, even at relatively low agonist concentrations. Thus, ATP both increases the potency of orthosteric agonists at class B GPCRs and reduces latency for adenylyl cyclase activation. SIGNIFICANCE STATEMENT: In addition to acting as a positive modulator of PTH1R signaling, extracellular ATP increases the potency of orthosteric agonists at other class B GPCRs and reduces the latency for adenylyl cyclase activation. Further insight into the precise mechanism of ATP-mediated potentiation of class B GPCR signaling may identify new targets for the development of therapeutic agents aimed at the treatment of endocrine disorders.Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.