In the epididymis a series of complex, sequential events transform immature, spermatozoa into mature, motile sperm with fertilizing ability. These events are not intrinsic to germ cells but rather are a direct result of exposure to, and interaction with, the environment created by the epididymal epithelium. Regional differences along the epididymis are essential in the establishment of the environment required for sperm maturation. Although parts of this process have been identified, the molecular basis for the segment-specific differences and how they contribute to the process of sperm maturation, are not yet resolved. The identification of genes expressed in a region-specific manner will provide valuable insight into the functional differences between the regions. To characterize gene expression in the different regions of the epididymis, microarrays containing 1176 rat cDNAs were used to examine gene expression in the initial segment, caput, corpus, and cauda epididymidis of the adult Brown Norway rat. Overall, the cauda epididymidis expressed the most genes and the corpus epididymidis the fewest. A small percentage of genes (3%) were expressed highly (greater than fivefold the average expression on the array) along the tissue. Segment-specific gene expression for genes expressed at high levels was observed in all epididymal segments except the corpus epididymidis. Of the genes on the array, 36% were expressed in all four epididymal segments; expression changes that were a minimum of twofold in either direction between adjacent segments are discussed. The expression of cathepsins and oxidative stress-related genes was investigated. Six of the eight cathepsins on the array (B, C, E, H, L, and K) were expressed above twofold background and showed different levels of expression along the duct with cathepsin K showing the most dramatic change (i.e., a decrease of 87% between the initial segment and the corpus epididymidis). There was also differential expression along the epididymis of many genes associated with oxidative stress defenses. Using the power of expression array technology, we have identified novel transcripts expressed in a segment-specific manner and been able to assess how the expression of several selected gene families is modulated along the epididymis.