Chronic treatment of mice with morphine affects the proliferation, differentiation, and function of immune cells. In the present study, we investigated the mechanism by which morphine inhibits phytohemagglutinin (PHA)/interleukln-1 (IL-1)-induced thymocyte proliferation. When compared to control cultures, morphinetreated thymocytes showed decreased steady-state levels of bioactive IL-2 and IL-2 mRNA. The reduced IL-2 concentration and reduced transcript levels correlated well with a decreased rate of synthesis of IL-2 mRNA as determined by nuclear runoff assays. Subsequent studies showed that morphine treatment affected transcriptional control elements of the IL-2 promoter by inhibiting the synthesis of a specific trans-activating nuclear factor, c-Fos. c-Fos mRNA levels measured by semiquantitative RT-PCR were significantly decreased in thymocytes following treatment with morphine and activation with PHA and IL-1. Under identical conditions, c-Jun mRNA levels were not altered. Electrophoretic mobility shift studies with the AP- 1 consensus oligonucleotide showed significantly decreased levels of AP-1- protein complex formation in nuclear extracts prepared from morphine-treated cells. These studies demonstrate for the first time that opioid alkaloids such as morphine can impair mitogen-lymphokine-activated thymocyte proliferation by interfering with transcriptional activation of the IL-2 gene.