Objective: To study expression of dysadherin in human testis, epididymis, and spermatozoa. Design: Prospective study. Setting: Basic research laboratory. Patient(s): Testis, epididymis, and testicular spermatozoa from patients under treatment and semen from volunteer donors. Intervention(s): Reverse transcription-polymerase chain reaction, immunohistochemistry, immunocytochemistry, and Western immunoblotting. Main Outcome Measure(s): Dysadherin messenger RNA (mRNA) analysis in testis, epididymis, and ejaculated spermatozoa, immunohistochemistry of both tissues, Western immunoblotting of tissue/cell extracts, and immunocytochemistry of spermatozoa. Result(s): Dysadherin mRNA was found in testis, epididymis, and ejaculated spermatozoa. Whereas testis and spermatozoa exhibited a distinctive 91-kDa protein form, the epididymis showed a 50-kDa moiety, also found in MDA-MB-231 breast cancer cells. Nucleotide sequence analysis revealed >99% homology between testicular and somatic cell mRNA, suggesting differential protein glycosylation. Dysadherin was immunodetected in round spermatids and testicular/ejaculated spermatozoa. It localizes to the acrosomal region and flagellum and colocalized with E-cadherin in the head and with the Na+,K+-ATPase alpha 4 subunit in the flagellum. Conclusion(s): This is the first report on expression of dysadherin in the male gonad and in spermatozoa. Its colocalization with E-cadherin and Na+, K+-ATPase leads us to postulate a role for dysadherin as a modulator of sperm function. (Fertil Steril (R) 2011;96:554-61. (C) 2011 by American Society for Reproductive Medicine.)