The fumaramate derivative of naltrexone, beta-funaltrexamine (beta-FNA), is a highly selective long-lasting mu opioid receptor antagonist that is active both in vitro and in vivo, presumably as a result of covalent binding to a mu receptor-based sulfhydryl group. Glutathione, which occurs in significant levels in brain and liver, was found to undergo a Michael-type reaction with beta-FNA in the test tube to give a stable conjugate 3 which occurred as an isomeric mixture. When tested in the GPI and MVD smooth muscle preparations, 3 was found to possess one-tenth the agonist activity of beta-FNA is both tissues, but showed no irreversible antagonist activity. The same result was found for the cysteine conjugate 4, except for some irreversible antagonism in the MVD. Both conjugates antagonize the antinociceptive effect of morphine in the mouse radiant heat tail-flick assay on icv administration. This antagonism persisted and actually increased over 24 h and generally paralleled the duration profile of beta-FNA. On sc administration, beta-FNA and 3 showed similar duration of antagonistic effect, while 4 exhibited only marginal activity at the early time interval. When the compounds are compared by the dose to produce equivalent antagonism, beta-FNA and 3 appeared equally effective and accessible by either route, whereas 4 showed a large difference between the two routes. It is possible that the ultra-long antagonism of the conjugates may result from their enzymatic conversion to beta-FNA in the central nervous system in view of the fact that conjugate 5, which cannot be converted to beta-FNA, did not produce antagonism of long duration in vivo. Alternatively, the protracted antagonism could arise from sequestration of 3 and 4 in tissue compartments that interface with mu opioid receptors.