Histochemical and biochemical studies suggest that the functions of the intermediate filament (IF) binding protein plectin comprise the physical linkage of IFs to each other and to other cytoskeletal elements, and their anchorage at membrane-attached junctional complexes. To further evaluate this hypothesis the expression, cellular distribution, and ultrastructure of plectin arrays were studied in rat glioma C6 cell subclones differing in IF protein (vimentin) expression. Here we show that plectin is expressed in a vimentin-negative C6 cell subclone (C6-D10) at levels similar to those of the vimentin-positive control subclone C6-D8. However, the amount of cytoskeleton-associated plectin found after extraction of cells with Triton X-100 or Triton X-100/high salt was significantly reduced in IF-negative compared to IF-positive cells. Using immunofluorescence microscopy, plectin structures were detected throughout the cytoplasm of IF-deficient cells. Unlike in IF-containing cells, where plectin colocalized largely with the vimentin network, in the IF-negative subclone the protein was mainly associated with polymeric actin structures. The release of plectin from IF-deficient cytoskeletons upon treatment with heavy meromyosin argued for specificity of the plectin microfilament interaction. Whole mount electron microscopy in conjunction with immunogold labeling of cytoskeletons revealed that in both IF-positive and IF-negative cells, plectin label specifically associated with thin (3-nm) filamentous structures that were clearly distinct from the major cytoskeletal filament systems. In IF-containing cells these filaments were found to link IFs to actin filaments and to connect vimentin filaments to each other. In IF-deficient cells, filamentous plectin structures were found to form dense cytoplasmic networks together with actin filaments and actin filament bundles. These data support the hypothesis that filamentous plectin arrays play an important role in the structural organization and mechanical integration of the cytoskeleton, in particular IFs and microfilaments.