The accessory structures (fibrous sheath and outer dense fibers) of the mouse sperm flagellum are composed of a heterogeneous array of polypeptides, including a prominent 82,000 M(r) fibrous sheath protein (p82). Using an antiserum against p82, indirect immunofluorescence showed specific reactivity to the entire length of the principal piece of the tail, indicating that p82 was localized to the fibrous sheath. Immunoblot analysis showed that while p82 was present in whole sperm and was highly enriched in the tail fraction, it was not detected in extracts of round and condensing spermatids, the developmental period when the fibrous sheath is assembled. Rather, a band of approximately 97,000 M(r) was recognized in these cells, suggesting that p82 was synthesized as a precursor. A full-length cDNA clone was isolated from a mouse mixed germ cell cDNA expression library following screening with anti-p82. A comparison of amino acid sequences from tryptic peptides and the N-terminus of purified p82 and the deduced amino acid sequence from the clone confirmed that this clone encoded p82. The sequence predicted that p82 has a molecular weight of approximately 72,000 and was synthesized as a precursor of approximately 92,000 M(r). Interestingly, while p82 was a unique spermatid-specific polypeptide, it has regional homologies to those domains within the A-Kinase Anchoring Protein group of polypeptides that are responsible for anchoring protein kinase A (PK-A) to the cytoskeleton. Furthermore, p82 specifically bound the regulatory subunit of PK-A when examined by a ligand blotting assay. These results suggest that p82 acts as a scaffolding protein for the subcellular localization of regulatory proteins in the flagellum.