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Outer dense fibres (ODF) are important substructures of mammalian sperm tails that are involved in the regulation of sperm motility. In this study, we investigated the identity of several sodium dodecyl sulphate (SDS)-insoluble ODF proteins. Bovine ODF were purified by separating sperm heads and tails using ultrasound and Percoll(R) density gradient centrifugation. Sperm flagella were treated with the detergent cetyltrimethylammonium bromide (CTAB). CTAB-insoluble material, which reportedly represents the ODF fraction, was collected, and electron microscopy confirmed a highly purified ODF fraction. We found after solubilization of this fraction with SDS that high amounts of insoluble material were retained after centrifugation. SDS-insoluble material was collected and quantitatively dissolved in 8 M urea. SDS-gel electrophoresis in the presence of urea revealed polypeptides with apparent molecular masses of approximately 25, 43, and 50 kDa. Subsequent immunoblotting with anti-cytokeratin antibodies detected two urea-soluble, SDS-insoluble proteins with apparent molecular masses of 45 and 66 kDa. The 45-kDa protein was identified as cytokeratin 19. An antibody reacting with a palette of cytokeratins (CK 1-18 and CK 20), KL1, was the only antibody that reacted with the 66-kDa polypeptide. We conclude that sperm ODF fractions contain at least one each of type I and type II intermediate filaments. As keratins and intermediate filaments are described as rope-like structures, we suggest that these intermediate filaments play an important structural or tension-bearing role in sperm flagella.