We investigated the relative importance of heroin and its metabolites in eliciting a behavioral response in mice by studying the relationship between concentrations of heroin, 6-monoacetylmorphine (6MAM), and morphine in brain tissue and the effects on locomotor activity. Low doses (subcutaneous) of heroin (< or =5 micromol/kg) or 6MAM (< or =15 micromol/kg) made the mice run significantly more than mice given equimolar doses of morphine. There were no differences in the response between heroin and 6MAM, although we observed a shift to the left of the dose-response curve for the maximal response of heroin. The behavioral responses were abolished by pretreatment with 1 mg/kg naltrexone. Heroin was detected in brain tissue after injection, but the levels were low and its presence too short-lived to be responsible for the behavioral response observed. The concentration of 6MAM in brain tissue increased shortly after administration of both heroin and 6MAM and the concentration changes during the first hour roughly reflected the changes in locomotor activity. Both the maximal and the total concentration of 6MAM were higher after administration of heroin than after administration of 6MAM itself. The morphine concentration increased slowly after injection and could not explain the immediate behavioral response. In summary, the locomotor activity response after injection of heroin was mediated by 6MAM, which increased shortly after administration. Heroin acted as an effective prodrug. The concentration of morphine was too low to stimulate the immediate response observed but might have an effect on the later part of the heroin-induced behavioral response curve.