A proteomic approach was used in this study to follow the protein expression of epididymal cells during the different phases of a cell culture protocol which was able to obtain an epididymal cell monolayer. The secretory activity of intact proximal and middle caput epididymal fragments and caput, corpus and cauda epithelial cell monolayers was examined on different days of culture. Transcriptomic activity was also followed by RT-PCR for the mRNA of several previously identified major proteins. During the establishment of epididymal cell cultures, a progressive shift was found in the pattern of protein secretion. The normal epididymal protein profile, specific for each epididymal region, was progressively replaced by a less specific profile with the secretion of new proteins. A correlation between protein secretion and the presence of the mRNA of the marker proteins was observed only in the first phase of culture. Most of the new proteins which appeared were characteristic of the secretion of cell monolayers cultivated over several weeks. Despite the significant modifications of the epididymal cell secretome, the presence of new proteins secreted only by cell cultures originating from a specific epididymal region shows the presence of remaining endogenous differentiation.