Previous studies have shown morphine (50.0 mg/kg, s.c.) suppresses cytotoxic T lymphocyte (CTL) activity in alloimmunized mice. The present study was undertaken to assess the dose-dependent nature of the suppressive effect of subchronic morphine exposure on CTL activity in vivo. In alloimmunized ICR mice, morphine (10.0-100.0 mg/kg, s.c.) administered daily for 5 days significantly suppressed CTL activity ( > 50%) in a dose-dependent and naltrexone (10.0 mg/kg, s.c.)-reversible fashion. The morphine-treated mice showed tolerance to the drug after daily exposure over 5 days as determined by measuring tail flick latency during thermal application. CD4(+)- and CD8(+)-depletion studies showed that effector cells were predominately CD8+ and mediated cytolysis through a Ca(++)-dependent pathway. Splenocytes obtained from the morphine (56.0-100.0 mg/kg)-treated, alloimmunized mice produced significantly less (30-40%) interferon-gamma but not interleukin-2 or interleukin-10 after antigen stimulation but not concanavalin A stimulation. Furthermore, this effect was dose- and time-dependent. In addition, there was a modest decrease (10%) in the interferon-gamma transcript level from the morphine (56.0-100.0 mg/kg)-treated, alloimmunized mice compared with vehicle-treated, alloimmunized mice as determined by quantitative reverse transcription-polymerase chain reaction. However, there were no differences in the perforin transcript levels between morphine- and vehicle-treated mice. Collectively, these results suggest that subchronic morphine exposure suppresses CTL activity in tolerized, alloimmunized mice through a pathway that may peripherally involve interferon-gamma production.