Epididymis and vas deferens form part of the male internal genital tract, and are dependent on androgens for their growth and development. To better understand the molecular action of androgens during male genital tract development, protein expression profiles were generated using 2-dimensional (2D) gels, for rat epididymides and vasa deferentia isolated on embryonic days (E) 17-21. Proteins that were differentially expressed between E17 and E21 were cut from the gels, digested into tryptic peptides and analyzed on a MALDI-TOF mass spectrometer. Using this approach, 20 proteins could be identified that were regulated in time and were categorized into cytoskeletal proteins, nuclear proteins, transport proteins, chaperones, and enzymes (mainly glycolytic). Furthermore, epididymides and vasa deferentia isolated on E19 were cultured in vitro in the absence or presence of 10 nM of the synthetic androgen R1881, for 9, 24, and 48 h. Under these conditions, regulation and post-translational modification were observed for glyceraldehyde 3-phosphate dehydrogenase, triosephosphate isomerase, hnRNP A2/B1 and hnRNP A3, similar to the observed changes in vivo. In addition, post-translational modification of RhoGDI1 (also named RhoGDI) was found in response to androgen. Androgen-induced post-translational modification of RhoGDI1 and glycolytic enzymes may be an important functional link between signaling pathways and cytoskeletal rearrangements in control of growth and development of the male internal genital tract.