The peripheral-type benzodiazepine receptor (PBR) is involved in many cellular functions, including steroidogenesis, oxidative processes, cellular proliferation, and apoptosis. Secretory and glandular tissues, especially steroid hormone-producing cells, are particularly rich in PBR. To understand the mechanisms of PBR expression and regulation, we established an mRNA expression profile in mouse tissues and cell lines and subsequently mapped the transcription start site and characterized the promoter of the gene. Our findings indicate that PBR tissue mRNA levels are relatively high in kidney, spleen, muscle, lung, adrenal gland, thymus, and stomach; are intermediate in pancreas, uterus, prostate, heart, and testis; and are low in brain and liver. Relatively high levels of PBR mRNA were also observed in the steroid-synthesizing MA-10 mouse Leydig tumor cells compared with adrenocortical Y1 mouse cells and nonsteroidogenic NIH-3T3 mouse fibroblasts, although PBR protein levels were much higher in both steroidogenic cells compared with fibroblasts. Transcription was initiated primarily at an adenine nucleotide 61 nucleotides upstream of the translation start site, but internal initiation was also observed. A 2.7-kb fragment of the mouse PBR promoter was cloned and sequenced. Sequence analysis revealed the absence of TATA or CCAAT boxes, but the presence of many putative transcription factor-binding sites, including Sp1/Sp3, AP2, Ik2, AP1, SOX, GATA, and SRY. Functional characterization revealed that two Sp1/Sp3 sites in the proximal promoter are important for basal activity in all cell lines tested and that the steroidogenic MA-10 and Y1 cells use different areas of the promoter compared with nonsteroidogenic NIH-3T3 cells.