Project Summary/Abstract This resubmitted R21 proposal is in response to the Notice of Special Interest (NOSI): Xylazine: Understanding Its Use and Consequences, NOT-DA-24-012. The goal of this study is to determine if xylazine and its metabolites act as agonists or positive allosteric modulators (PAMs) at the mu and kappa opioid receptors (MOR, KOR). Additional studies will determine if xylazine and its metabolites are agonists, antagonists, or inverse agonists at the sigma 1 (s1R) and sigma 2 receptors (s2R). Xylazine binds with nM affinity to the sigma receptors. Illegal drugs, particularly fentanyl and stimulants, are being mixed with xylazine to enhance drugs effects or increase street value by increasing weight. Xylazine is not approved for human use. Xylazine is historically regarded as an α2-adrenergic receptor agonist based on physiological studies using α2-adrenergic receptor antagonists. However, a recent studies showed that the opioid antagonist naloxone induced withdrawal from xylazine in mice and xylazine had micromolar affinity for the α2-adrenergic receptor. Our preliminary data shows that xylazine had a Ki value of 330 ± 13 nM for inhibiting binding to the human KOR. Xylazine also inhibited forskolin-induced cyclic AMP levels in CHO cells expressing the human KOR and stimulated G protein activity as measured by a BRET assay. The following Specific Aims will characterize the potency and pharmacological properties of xylazine. Aim 1 will determine if xylazine and its metabolites act as PAMs at the MOR and KOR as shown with G protein and β-arrestin signaling. Bias signaling through the six Gαi/o/z proteins will be determined and compared with results obtained with β-arrestin recruitment. In addition, experiments will determine if xylazine and its metabolites inhibit forskolin-stimulated cAMP as an agonist or as a PAM. Radioligand binding experiments will determine if xylazine acts as a PAM by increasing the affinity of agonists such as fentanyl for the