Previous studies in our laboratory have documented the occurrence of naloxone-precipitated opioid abstinence from 45 minutes to 6 hours after acute morphine administration in humans. This study extended the morphine-naloxone interval to 24 hours and examined the effect of repeated naloxone challenges on withdrawal responses. Six male nondependent opiate users participated in eight experimental sessions in which they received single IM injections of morphine (18 mg/70 kg) followed 6 and 24 hours later by challenge sessions with IM placebo or naloxone (10 mg/70 kg). Naloxone challenge at 6 hours postmorphine reversed morphine-induced miosis and subjective reports of opiate symptoms, drug high, good drug effects, and drug liking. At 24 hours postmorphine, naloxone had no effect on these measures, which had returned to premorphine levels. However, at 6 and 24 hours postmorphine, naloxone precipitated subjective symptoms and observer-rated signs of opioid abstinence. When naloxone challenge at 24 hours was preceded by naloxone at 6 hours postmorphine, the magnitude of abstinence symptoms and signs was attenuated. These data suggest that morphine-induced adaptational changes underlying the development of physical dependence persist beyond other measureable agonist effects, and that these changes are disrupted or reversed by repeated antagonist administration.