The relative affinity to opiate receptors of heroin, 6-acetylmorphine and morphine was estimated by determining their ability to displace specifically bound ³H-naltrexone from rat brain opiate binding sites. In vitro hydrolysis of heroin to 6-acetylmorphine was monitored in the binding assay filtrate by use of a quantitative HPLC procedure. The rate of heroin hydrolysis was significantly slower at 0°C than at 37°C. The displacement of 1 nM ³H-naltrexone by unlabeled ligand at concentrations ranging from 7 to 500 nM was measured at 0°C for 120 minutes, yielding IC₅₀ values of heroin = 483 nM, 6-acetylmorphine = 73 nM and morphine = 53 nM. When the binding data for heroin were recalculated to include the displacement that could be attributed to the 6-acetylmorphine derived from heroin degradation during the incubation, all of the apparent heroin binding was accounted for by the 6-acetylmorphine. These results are consistent with previous reports of the low binding affinity of morphine congeners (e.g., codeine) that lack a free phenolic 3-hydroxyl group and support the view that heroin is a prodrug which serves to determine the distribution of its intrinsically active metabolites, 6-acetylmorphine and morphine.