A murine epididymal retinoic-acid-binding protein (mE-RABP) is specifically expressed in the mid/distal caput epididymidis and is androgen regulated. The murine epididymal protein of 17 kDa (mEP17) gene, a novel gene homologous to mE-RABP, is located within 5 kb of the 5′-flanking region of the mE-RABP gene. In contrast, expression of the mEP17 gene is restricted to the initial segment and regulated by factor(s) contained in testicular fluid. To identify cis-DNA regulatory element(s) involved in the tissue- and region-specific expression of the mEP17 gene in transgenic mice, we have studied the expression of a transgene containing 5.3 kb of the 5′-flanking region of the mEP17 gene (5.3mEP17) linked to chloramphenicol acetyltransferase (CAT) reporter gene. Significant caput epididymidis-specific CAT activity was detected in transgenic mouse lines; and CAT gene expression is restricted to the initial segment, as is the expression of the endogenous mEP17 gene. Ontogenic expression and testicular factor dependency also mimic that of endogenous mEP17 gene. These results suggest that the 5.3mEP17 fragment contains all the information required for spatial and temporal expression in the mouse epididymis. The 5.3mEP17 fragment will be useful to express a foreign gene of interest in the epididymis in an initial segment-specific manner.