Purpose: To compare the transcriptome profiles (RNA-seq) of cultured human epididymis cells and tissue from the caput, corpus and cauda regions of the human epididymis. Methods: Human epididymis tissue was obtained with Institutional Review Board approval from 3 patients (UC05, UC06, UC09, range: 22 - 36 years) undergoing inguinal radical orchiectomy for a clinical diagnosis of testicular cancer. None of the epididymides had extension of the testicular cancer. The three anatomical regions: caput, corpus and cauda, were separated and segments of each snap frozen. Adult human epididymis epithelial (HEE) cultures were also established from tissue. RNA was extracted from both tissue and cultured HEE cells and RNA-seq libraries prepared (TruSeq RNA Sample Preparation Kit v2, Low-Throughput protocol, Illumina). Libraries were sequenced on Illumina HiSeq2500 machines. Data were analyzed using TopHat and Cufflinks. Results: Libraries generated ~19-39 million reads per library from the cells (95-99% mapping to the human genome) and ~14-39 million reads from the tissue samples (84-99% mapped). Raw reads were aligned to the genome with Tophat and gene expression values were processed using Cufflinks as Fragments Per Kilobase per Million mapped fragments (FPKM). FPKM values were subject to principle component analysis, which revealed that though caput, corpus and cauda cell samples respectively from UC05, UC06 and UC09 clustered together. RNA-seq data from the 3 biological replicas (UC05, UC06 and UC09) of caput, corpus and cauda were pooled for further analysis. Cufflinks was used to determine differentially expressed genes (DEGs) between caput, corpus and cauda cells, combined from the 3 donors. The gene expression profiles of corpus and cauda are remarkably similar and both differ from the caput to a similar degree. We identified ~40 genes differentially expressed between corpus and cauda and more than 1600 DEGs between caput and cauda. The DEGs for each comparison (caput and corpus/cauda) were analysed using a gene ontology process enrichment analysis (DAVID, Huang et al., NAR 2009;37:1-13, Huang et al., 2009 Nat Prot 4:44-57). Conclusions: Here we describe an in depth analysis of the gene expression repertoire of primary cultures of epithelial cells and intact tissues from each region of the adult human epididymis. These data will be valuable to decipher pathways of normal epididymis function and aspects of epididymis disease that cause male infertility. Overall design: RNA-seq was performed on libraries generated from caput, corpus and cauda-derived cultured cells (passage 2 or 3) from 3 donors and on caput, corpus and cauda tissue from 2 of the same donors. Donor age range: 22 - 36 years.