© 2015 The Authors. Background: Opioid analgesics are frequently prescribed for chronic pain. One expected consequence of long-term opioid use is the development of physical dependence. Although previous resting state functional magnetic resonance imaging (fMRI) studies have demonstrated signal changes in reward-associated areas following morphine administration, the effects of acute withdrawal on the human brain have been less well-investigated. In an earlier study by our laboratory, ondansetron was shown to be effective in preventing symptoms associated with opioid withdrawal. The purpose of this current study was to characterize neural activity associated with acute opioid withdrawal and examine whether these changes are modified by ondansetron. Methods: Ten participants were enrolled in this placebo-controlled, randomized, double-blind, crossover study and attended three acute opioid withdrawal sessions. Participants received either placebo or ondansetron (8. Ymg IV) before morphine administration (10. Ymg/70. Ykg IV). Participants then underwent acute naloxone-precipitated withdrawal during a resting state fMRI scan. Objective and subjective opioid withdrawal symptoms were assessed. Results: Imaging results showed that naloxone-precipitated opioid withdrawal was associated with increased neural activity in several reward processing regions, including the right pregenual cingulate, putamen, and bilateral caudate, and decreased neural activity in networks involved in sensorimotor integration. Ondansetron pretreatment did not have a significant effect on the imaging correlates of opioid withdrawal. Conclusions: This study presents a preliminary investigation of the regional changes in neural activity during acute opioid withdrawal. The fMRI acute opioid withdrawal model may serve as a tool for studying opioid dependence and withdrawal in human participants.