Several lipocalins are present in the mouse epididymis and are thought to play a role in sperm maturation by transporting lipophilic molecules. We have previously reported that two lipocalin genes, mERABP (mouse epididymal retinoic acid binding protein), and mEP17 (mouse epididymal protein of 17 kDa), derived from an ancestral gene, are specifically expressed in the epididymis. In the present study, a polyclonal antibody was raised against a recombinant protein to investigate the presence and the regulation of mEP17. mEP17 was detected in the supranuclear region of the principal cells of the initial segment, the clear cells of the caput epididymidis, and the lumen of the mid/distal caput but not of the distal epididymis. Initial segment and caput tissue extracts were subjected to HPLC separation. After electrophoresis of the immunoreactive mEP17-enriched fractions, the immunoreactive band was analyzed by mass spectrometry to identified mEP17 unambiguously. After two-dimensional electrophoresis, mEP17 appeared as a train of five 22-kDa spots with a range of pI (isoelectric point) from 5.8-6.7. N-glycanase digestion gave rise to a single spot of 17 kDa and pI 6, the predicted mass and pI. During ontogeny, mEP17 was detected as early as 3 wk of age and increased afterward. After bilateral orchiectomy, mEP17 disappeared 2 d after surgery and was not restored after testosterone replacement. After unilateral orchiectomy, mEP17 levels decreased only in the orchiectomized side. After cryptorchidism or busulfan treatment, mEP17 levels were either greatly diminished or not detected. This suggests that mEP17 is dependent on testicular factor(s) that may have a germ cell origin. Altogether, our data demonstrate that mEP17 spatial expression, regulation, and fate are different from that of the highly related mouse epididymal retinoic acid binding protein. This suggests that these two related proteins exhibit distinct functions in the mouse epididymis.