We previously reported that the 34-bp cis-acting element of the mouse μ opioid receptor (MOR) gene represses transcription of the MOR gene from the distal promoter. Using a yeast one-hybrid screen to identify potential transcription factors of the MOR promoter, we have identified PU.1 as one of the candidate genes. PU.1 is a member of the ets family of transcription factors, expressed predominantly in hematopoietic cells and microglia of brain. PU.1 plays an essential role in the development of both lymphoid and myeloid lineages. Opioids exert neuromodulatory as well as immunomodulatory effects, which are transduced by MOR. Moreover, MOR-deficient mice exhibit increased proliferation of hematopoietic cells, suggesting a possible link between the opioid system and hematopoietic development. The PU.1 protein binds to the 34-bp element of the MOR gene in a sequence-specific manner confirmed by electrophoretic mobility shift assay and supershift assays. We have also determined endogenous PU.1 interactions with the 34-bp element of MOR promoter by chromatin immunoprecipitation assays. In co-transfection studies PU.1 represses MOR promoter reporter constructs through its PU.1 binding site. When the PU.1 gene is disrupted as in PU.1 knock-out mice and using small interfering RNA-based strategy in RAW264.7 cells, the transcription of the endogenous target MOR gene is increased significantly. This increase is probably mediated through modification of the chromatin structure, as suggested by the reversal of the PU.1-mediated repression of MOR promoter activity after trichostatin A treatment in neuroblastoma NMB cells. Our results suggest that PU.1 may be an important regulator of the MOR gene, particularly in brain and immune cells.