Clusterin is a ubiquitous glycoprotein that is promiscuously expressed at a low basal level but can be highly induced by a variety of stress conditions. In contrast, in some secretory cells associated with tissue-fluid interfaces such as the Sertoli cells in the testis, clusterin demonstrates high constitutive expression. In this study, we address the mechanisms that regulate the constitutive expression of the clusterin gene by using primary cultures of immature rat Sertoli cells. We have identified a region of the rat clusterin gene promoter that activated transcription only in Sertoli cells and that mapped between positions −426 and −311. Sequence analysis of this region revealed a high concentration of potential regulatory elements. Using gel-shift assays combined with hydroxyl radical footprinting, we identified the elements recognized by the Sertoli cell nuclear factors. Comparison of the interactions with this region of the nuclear factors from different cell types demonstrated that recognition of the core-enhancer element is specific for the Sertoli cells, and in vitro, the core region was recognized by the transcription factor CBF. Transient transfections showed that a core enhancer is responsible for more than a half of the total promoter activity and is an essential element for the cell-specific activity of the Sertoli-specific region. In addition to the core enhancer, tandem Sp1 sites are also required for maximal activity of this region.