IFAP-300K is a 300,000-mol-wt intermediate filament-associated protein previously identified in the baby hamster kidney fibroblastic cell line (BHK-21) by a monoclonal antibody (Yang H.-Y., N. Lieska, A. E. Goldman, and R. D. Goldman, 1985, J. Cell Biol., 100: 620-631). In the present study, this molecule was purified from the high salt/detergent-insoluble cytoskeletal preparation of these cells. Gel filtration on Sephacryl S-400 in the presence of 7.2 M urea allowed separation of the high molecular weight fraction from the structural intermediate filament (IF) subunits desmin and vimentin, designated 54K and 55K, respectively, and other low molecular weight polypeptides. DE-52 cellulose chromatography of the high molecular weight fraction using a linear NaCl gradient in 8 M urea yielded a pure 300,000-mol-wt species which was confirmed to be IFAP-300K by immunological and peptide mapping criteria. Two-dimensional PAGE of native BHK IF preparations followed by immunoblot analysis demonstrated the inability of the IFAP-300K-immunoreactive material to enter the first dimensional gel except as a 200,000-mol-wt doublet which presumably represented a major proteolytic derivative of IFAP-300K. The molecule's pl of 5.35, as determined by chromatofocusing, and its amino acid composition were extremely similar to those of BHK cell vimentin/desmin despite their non-identity. Ultrastructurally, IFAP-300K preparations in low salt buffers existed as particles composed of one or two elliptical units measuring 16 X 20 nm. In physiological salt buffers, the predominant entities were large, elongated aggregates of the elliptical units, which were able to be decorated by using the immunogold technique with monoclonal anti-IFAP-300K. Compared with the morphology of homopolymer vimentin IF, in vitro recombination studies using column-purified vimentin and IFAP-300K demonstrated the additional presence of aggregates similar in appearance to IFAP-300K at points of contact between IFs. Antibody decoration and immunogold labeling of these recombined preparations using rabbit antidesmin/vimentin and monoclonal anti-IFAP-300K confirmed the identity of the inter-filament, amorphous material as IFAP-300K. The presence of IFAP-300K at many points of intersection and lateral contact between IFs, as well as at apparent inter-filament "bridges," in these recombined specimens was identical to that seen both in situ and in native IF preparations. No such co-sedimentation was found in vitro between actin and IFAP-300K. No effects of IFAP-300K upon the kinetics of IF polymerization were detected by turbidimetric measurements.