The extracellular-matrix glycoprotein, tenascin, consists of disulfide-linked subunits of 190, 200 and 230 kDa (the three splicing variants reported in chicken) and usually exists as a six-armed structure under the electron microscope. We used monoclonal antibodies to isolate and characterize different splicing variants and proteolytic fragments obtained from the native protein. Purified monomeric tenascin has a native molecular mass of 216 kDa and is structured as single arms. Tenascin fragments obtained by pepsin digestion bind to monoclonal antibody (mAb) TnM1 which is directed against epidermal-growth-factor-like repeats in the N-terminal half of all subunits. These fragments represent the thin proximal part of the tenascin arms and they are still partially linked to dimers and trimers via disulfide bridges. Using mAb Tn68, that reacts with a fibronectin-type-III repeat towards the C-terminus, a tenascin fragment, generated by treatment with pronase, can be isolated. Ultrastructurally, this fragment looks like the thicker distal part of the tenascin arms. Only the 230-kDa variant of tenascin gives rise to this distal fragment after cleavage within the alternatively spliced fibronectin-type-III repeats. Native tenascin and all fragments containing the distal part of its arms bind to heparin-agarose, whereas the proximal fragments do not. Oligomeric and monomeric tenascin inhibit fibronectin-mediated fibroblast spreading with comparable efficiency when added to the culture medium, while the proximal fragment has no effect. The distal fragment as well as reduced and alkylated tenascin are active in this assay, but only at higher molar concentrations when compared to the native protein.