Tektins are evolutionarily conserved filament-forming proteins localized in flagella and cilia, and have been reported to be involved in the stability and structural complexity of axonemal microtubules. Five mammalian Tektins (Tektin1–5) have been reported. Of these, Tektin2 (TEKT2) has been found to be required for normal flagellum structure and function. Tekt2-null sperm display flagellum bending and reduced motility, probably due to disruption of the dynein inner arm. However, the subcellular localization of TEKT2 in spermatozoa has not been clarified at the ultrastructural level. To elucidate the molecular localization of TEKT2 in flagella of rat spermatozoa, we performed confocal laser scanning microscopy, extraction of flagella followed by immunoblot analysis, and immunogold electron microscopy. Extraction of sperm flagella by SDS-EDTA resulted in complete extraction of axonemal tubulins, while TEKT2 was only partially released from flagella, suggesting that TEKT2 might be present in the peri-axonemal component, not directly associated with axonemal tubulins. Confocal laser scanning microscopy and pre-embedding immunoelectron microscopy revealed that TEKT2 is associated with the surface of outer dense fibers (ODFs). TEKT2 may function as an ODF-affiliated molecule required for flagellum stability and sperm motility.