Thimet oligopeptidase (TOP) is a thiol-dependent metallopeptidase, which can cleave and thereby modulate the activity of many neuropeptides. The enzyme is active in many endocrine tissues, including testis, brain, and pituitary. In rat, the richest source of TOP is the testes, with a specific activity fivefold that of brain. The mechanism whereby rat TOP expression is regulated at the transcriptional level has been examined by reporter gene assay and electromobility shift assays after isolation of 1020 bp of upstream sequence. Computer analysis predicts a number of potential transcription factor-binding sites, which were examined by deletion analysis and DNA-binding studies. The promoter or its deletion fragments were fused to luciferase reporter gene vectors and introduced into GH3 pituitary, COS-1 kidney, MAT-Lu prostate, and GC-2spd(ts) spermatid cells. Two regions of the promoter have been identified: a positively acting region (-901/-219) and a strong negatively acting region (-219/-102). Concomitantly, potential transcription factors interacting with the cis-acting elements of the promoter were studied by gel electromobility shift assays. This work has identified a number of transcription factor-binding sites. However, no differences in the binding behavior in the various cell lines was observed.