Multiple gamma-glutamyl transpeptidase (GGT) messenger RNAs (mRNAsII-IV) are expressed in a region-specific manner in the rat epididymis. In the present study, we examined the role(s) of plasma testosterone (T) and testicular factors in regulating the region-specific pattern and quantity of GGT mRNAs expressed along the epididymal duct. Northern blot and ribonuclease protection analyses showed that bilateral orchiectomy for 1, 5, and 15 days dramatically reduced the expression of GGT mRNAsII-IV in the initial segment. Expression of GGT mRNAII and mRNAIII was maintained in the initial segment of orchiectomized animals receiving T implants that maintain normal serum T concentrations, but GGT mRNAIV expression remained low relative to sham-operated control values. Unilateral efferent duct ligation decreased GGT mRNAIV expression only in the initial segment. Hence, expression of GGT mRNAIV in the initial segment was not maintained by circulating T and required a factor(s) of testicular origin. In caput epididymidis, expression of GGT mRNAII and mRNAIII declined after orchiectomy and was not completely restored to control values in orchiectomized animals by plasma T alone, but also required a testicular factor(s). In contrast to the initial segment, expression of GGT mRNAIV in the corpus and cauda epididymidis did not require T and/or a testicular factor(s), as expression of this transcript remained unchanged in these regions after 1, 5, and 15 days of orchiectomy, orchiectomy and T replacement, and after unilateral efferent duct ligation. In the cauda epididymidis, expression of GGT mRNAII required circulating androgens and was unaffected by unilateral efferent duct ligation, whereas GGT mRNAIII expression was repressed by T. These data demonstrate that circulating T and a factor(s) of testicular origin differentially regulate the expression of each GGT mRNA in a region-specific manner.