The disposition of morphine in rat brain and serum was determined over 48 h after s.c. doses. Free morphine was measured by a specific assay using 3H-labeling together with high-pressure liquid chromatography separation, with a sensitivity of 1 nM (0.3 ng morphine/ml). This study revealed the persistence of free morphine in nanomolar concentrations over at least 24 h after a single analgesic dose. The terminal half-life of morphine elimination was 5 h. Total radioactivity was retained in the body at much higher concentrations. Similar disposition of [C-1-3H]morphine and [N-14CH-3] morphine ruled out any major metabolic alterations at these positions, including N-demethylation. Irreversible binding to insoluble tissue components, which was previously linked to tolerance, was observed only to the extent of less than 20% of total tissue radioactivity and was not unique to brain tissue. The persistence of morphine and its metabolites may be related to protracted opiate effects, i.e., as withdrawal symptoms after addiction.