Acute opiate exposure produces a state of dependence in humans and animals, which is revealed by signs and symptoms of withdrawal precipitated by opioid receptor antagonists. The physiological changes that underlie this state of acute dependence develop rapidly and can persist long after the end of chronic opiate exposure. The purpose of this investigation was to determine the persistence of acute dependence after a single morphine exposure in rodents, focusing on changes in behavior thought to reflect the negative emotional consequences of withdrawal. The acoustic startle reflex and conditioned place aversion were measured following naloxone administration at different time points after a single morphine exposure. Naloxone administration produced significant potentiation of acoustic startle-a form of anxiety-like behavior-for at least 80 days after one exposure to morphine. In contrast, naloxone produced a conditioned place aversion 24 h but not 20 days after one morphine exposure. Together with existing literature, these results suggest acute as well as chronic opiate exposure leave rodents persistently vulnerable to express anxiety-like behavior in response to opioid receptor antagonists or stressful experience. The adaptations in brain function that underlie this protracted state of dependence may provide a foundation for the escalation of withdrawal severity that develops over repeated opiate exposure, and increase the likelihood of progression from casual drug use to compulsive drug abuse.