Abstract Opioid overdose deaths are the result of an on-target effect of compounds acting at the mu opioid receptor (MOR) causing severe respiratory depression. Fentanyl is a highly potent synthetic MOR agonist. Literature reports indicate the drastic rise in opioid-induced overdose deaths in the United States is due to the increased prevalence of fentanyl and its analogs (known as fentalogs) in street drugs. Indeed, more than 70% of opioid overdose deaths involve fentanyl or its analogs and a recent DEA press release stated that 2 in 5 counterfeit opioid medications contain a lethal dose of fentanyl. The current standard of care and the only medication currently available currently to treat opioid overdose is naloxone which was approved by the US Food and Drug Administration almost 50 years ago. However, reports suggest that fentalog-induced overdose is resistant to reversal by naloxone, which is not sufficiently potent, rapid, or long lasting with the result that overdose patients can re-narcoticize and are less likely to survive. In addition, there is evidence that successful resuscitation is further compromised because fentanyl produces unique physiological outcomes, for example wooden chest syndrome. Therefore, the is an urgent need for more effective therapies to reverse fentanyl-induced overdose. We propose the hypothesis that that an opioid antagonist that is developed from fentanyl and is therefore structurally related to fentanyl and binds in an analogous way to MOR, will reverse all aspects of opioid overdose caused by fentanyl and its illicit analogs. To this end we have identified fentanyl derivatives (R03 DA 048129 and R21 DA051723) that act as high affinity MOR antagonists both in vitro and in vivo. The objective of this proposal is to build on these initial leads to develop proprietary analogues, that have high affinity for MOR, and high antagonist potency together with favorable pharmacokinetic characteristics, i.e. rapid onset and with a length of action that prevents re-narcotization. Overall, this study could lead to the identification and development of an efficient reversal agent for fentanyl overdose.