The epididymal maturation of spermatozoa is regulated by changes in the luminal ion concentration and the processing of the sperm surface membrane by several glycosidases and proteases. In the present study, we identified five novel protease inhibitors that are highly expressed in the mouse epididymis. Four of the proteins were found to belong to the Kazal protease inhibitor family and were named SPINK8, SPINK10, SPINK11, and SPINK12, whereas one of the proteins, WFDC10, contained the WAP four-disulfide core domain structure. The novel genes showed very specific segmental expression patterns. The expression of all the five genes was regulated by testis-derived factors and decreased after gonadectomy. With the exception of Spink11, mRNA levels could be restored by testosterone replacement. We hypothesize that the protease inhibitors discovered represent a group of epididymal genes that contribute to the regulation of sperm maturation by regulating the proteolytic processing of the sperm membrane during epididymal transit.