OBJECTIVE: To establish cultures of epithelial cells from all regions of the human epididymis to provide reagents for molecular approaches to functional studies of this epithelium. DESIGN: Experimental laboratory study. SETTING: University research institute. PATIENT(S): Epididymis from seven patients undergoing orchiectomy for suspected testicular cancer without epididymal involvement. INTERVENTION(S): Human epididymis epithelial cells harvested from adult epididymis tissue. MAIN OUTCOME MEASURE(S): Establishment of a robust culture protocol for adult human epididymal epithelial cells. RESULT(S): Cultures of caput, corpus, and cauda epithelial cells were established from epididymis tissue of seven donors. Cells were passaged up to eight times and maintained differentiation markers. They were also cryopreserved and recovered successfully. Androgen receptor, clusterin, and cysteine-rich secretory protein 1 were expressed in cultured cells, as shown by means of immunofluorescence, Western blot, and quantitative reverse-transcription polymerase chain reaction (qRT-PCR). The distribution of other epididymis markers was also shown by means of qRT-PCR. Cultures developed transepithelial resistance (TER), which was androgen responsive in the caput but androgen insensitive in the corpus and cauda, where unstimulated TER values were much higher. CONCLUSION(S): The results demonstrate a robust in vitro culture system for differentiated epithelial cell types in the caput, corpus, and cauda of the human epididymis. These cells will be a valuable resource for molecular analysis of epididymis epithelial function, which has a pivotal role in male fertility.