Gamma-glutamyl transpeptidase (GGT) mRNA-IV is highly expressed in the initial segment of the rat epididymis and is regulated by testicular factors. The promoter region for GGT mRNA-IV contains five conserved polyomavirus enhancer activator 3 (PEA3)-binding motifs (5'-AGGAAG-3'). We hypothesize that PEA3 is present in the rat epididymis and is regulated by one or more testicular factors. Western blot analyses showed that a 62-kDa protein was detected in the nuclear extract from the rat initial segment at higher levels than in the distal epididymal regions. Electrophoretic mobility shift assays (EMSAs) showed that the nuclear extract specifically bound to the PEA3 motif, forming a DNA-protein complex. This complex contained the 62-kDa PEA3 protein as demonstrated by EMSAs and Southwestern analyses. Northern blot analyses and RNase protection analyses showed that PEA3 mRNA was predominantly expressed in the initial segment as compared to the distal epididymal regions and was under the regulation of testicular factors. These results suggest that PEA3 could be involved in the regulation of expression of the rat GGT mRNA-IV gene in response to testicular factors in the initial segment.