Juxtanuclear birefringent caps (FC) containing 10-nm filaments form during the early stages of baby hamster kidney (BHK-21) cell spreading. FC are isolated from spreading cells after replating by treatment with 0.6 M KC1, 1% Triton X-100 (Robin & Haas Co., Philadelphia, Pa.) and DNase I in phosphate-buffered saline. Purified FC are birefringent and retain the pattern of distribution of 10-rim filaments that is seen in situ. Up to 90% of the FC protein is resolved as two polypeptides of~ 54,000 and 55,000 molecular weight on sodium dodecyl sulfate (SDS) polyacrylamide gels. The protein is immunologically and biochemically distinct from tubulin as determined by indirect immunofluorescence, double immunodiffusion, one-dimensional peptide mapping by limited proteolysis in SDS gels, and amino acid analysis. The BHK-21 FC amino acid composition, however, is very similar to that obtained for 10-nm filament protein derived from other sources including brain and smooth muscle. Partial disassembly of 10-rim filaments has been achieved by treatment of FC with 6 mM sodium-potassium phosphate buffer, pH 7.4. The solubilized components assemble into distinct 10-nm filaments upon the addition of 0.171 M sodium chloride.