Vimentin intermediate filaments are a major cytoskeletal constituent of cells of mesenchymal origin. They have been colocalized with a variety of intracellular structures such as actin filaments and the plasma membrane. Labeled actin filaments, observed in vitro by fluorescence microscopy, break in the presence of polymerizing vimentin; the time course is consistent with stopped-flow measurements of vimentin polymerization. This breakage phenomenon appears to be specific for vimentin. Inhibition of vimentin network formation was observed with phosphatidyl inositol phosphate (PI(4)P) and phosphatidyl inositol bisphosphate (PI(4,5)P2), but not phosphatidyl choline (PC), phosphatidyl serine (PS), or phosphatidyl inositol (PI). Taken together, these results indicate a specific interaction of vimentin with F-actin and polyphosphoinositide lipids.