The mammalian epididymis is an exceptionally long ductal system tasked with the provision of one of the most complex intraluminal fluids found in any exocrine gland. This specialized milieu is continuously modified by the combined secretory and absorptive activity of the surrounding epithelium and thus finely tuned for its essential roles in promoting sperm maturation and storage. While considerable effort has been focused on defining the composition of the epididymal fluids, relatively less is known about the intracellular trafficking machinery that regulates this luminal environment. In this project we have characterized the ontogeny of expression of a master regulator of this machinery, namely the dynamin family of mechanoenzymes. Our results show that dynamin is expressed at high levels in the juvenile mouse epididymis suggesting it may hold an important role in the development of this organ. However, in adult mice dynamin takes on a heterogeneous pattern of expression such that the different isoforms displayed both cell and region specific localization. Specifically, dynamin 1 and 3 were predominately localized in the distal regions (corpus and cauda) of the epididymis where they were found within clear cells and principal cells, respectively. In contrast, dynamin 2 was found to be expressed throughout the epididymis, but localized to the Golgi apparatus of the principal cells in the proximal (caput) region and the luminal border of these cells in more distal regions. On the basis of these data we propose that different isoforms of the dynamin family contribute to the regulation of the epididymal milieu that ultimately promotes sperm maturation. In support of this hypothesis we were able to show that selective inhibition of dynamin in an immortalized mouse caput epididymal cell line was able to alter the profile of proteins secreted from these cells.