The testis expresses a variety of cadherin superfamily members including classic cadherins and protocadherins. This report describes the first localization of a protocadherin protein in testis and sperm. After cloning rat cDNAs for protocadherin α3 and α4, isoform-specific polyclonal antibodies were generated against protocadherin α3. Western blotting of rat testis showed that protocadherin α3 was solubilized completely by Triton X-100, in contrast to the adhesion junction components N-cadherin, β-catenin, and p120 catenin. Corroborating this data, protocadherin α3 was immunolocalized to the spermatid acrosomal area, intercellular bridge, and flagellum, but not classic cadherin-based adhesion junctions. Acrosome-associated protocadherin α3 was first detected at step 8 of spermiogenesis, and this association remained on cauda epididymal sperm. Acrosome immunostaining was reduced, but present, in acrosome-reacted sperm. Spermatid intercellular bridges became positive for protocadherin α3 coincident with the appearance of plectin, occurring at spermiogenic steps 8 to 9, and elongate spermatid bridges remained positive throughout spermatogenesis. The developing flagellum was uniformly immunostained for protocadherin α3 up to approximately spermiogenic step 17. Subsequently, flagellar immunostaining was confined to the principal piece, and this pattern continued in cauda epididymal sperm. These data show that protocadherin α3 performs functions unique from classic cadherins in spermatogenesis and suggest a role for protocadherin α3 in organizing germ cell-specific structures including the intercellular bridge, flagellum, and acrosome.