The negative motivational aspects of withdrawal include symptoms of both anxiety and depression, and emerge after termination of chronic drug use as well as after acute drug exposure. States of acute withdrawal are an inherent part of intermittent drug use in humans, but the contribution of acute withdrawal to the development of addiction has received limited systematic investigation, because of a lack of preclinical models for withdrawal states that emerge spontaneously after acute drug exposure. Here, we have characterized a spontaneous increase in the magnitude of the acoustic startle reflex (ie, spontaneous withdrawal-potentiated startle) that emerges after acute morphine administration in rats, and compared the time course of startle potentiation and place conditioning. We find that startle potentiation seems to be related to a decrease in opiate receptor occupancy and reflects an anxiety-like state with a pharmacological profile similar to other signs of opiate withdrawal. Spontaneous startle potentiation emerges before the rewarding effects of morphine have subsided, even though naloxone administration after a single morphine exposure causes both startle potentiation and conditioned place aversion (CPA). These results show that negative emotional signs of withdrawal develop after just one exposure to morphine, and are likely a recurrent aspect of intermittent drug use that may contribute to the earliest adaptations underlying the development of addiction. Furthermore, the dissociation between spontaneous startle potentiation and CPA suggests anxiogenic and dysphoric manifestations of opiate withdrawal may be mediated by distinct neural mechanisms that are progressively engaged as withdrawal unfolds.