The purpose of this study was to examine the possible effects of in vivo morphine (Mor) treatments on macrophage involvement in the mediation of reductions in T-cell functions. Male BALB/c mice were exposed to multiple sc injections of Mor in increasing doses twice a day for 4 days. T-lymphocyte proliferation in response to alloantigen was significantly affected by Mor, while Mor treatment failed to suppress PMA/A23187-induced macrophage-depleted lymphocyte proliferation. Interestingly, after separation of lymphocytes and macrophages from control or Mor-treated mice, Con A-stimulated T-cell proliferation was suppressed only when Mor-treated macrophages were combined with control or Mor-treated lymphocytes. Further, addition of exogenous interleukin (IL)-1 and IL-2 promoted the proliferative responses of lymphocytes obtained from Mor-treated mice to Con A. Studies of the mechanism of suppression show that macrophages are the primary target cells, since macrophage-depleted lymphocyte preparations produced little suppression, and that prostaglandin E2 and reactive oxygen intermediates (ROI) are important mediators possessing immunoinhibitory activities, since indomethacin or scavengers of ROI abrogated the suppression. These results demonstrate that Mor-triggered release of inhibitory macrophage metabolites and decrement in soluble cytokines production are involved in the immunosuppressive effects caused by subchronic in vivo repetitive administrations of the drug of abuse.