The major protein of intermediate-sized filaments in mouse 3T3 cells, for which the name vimentin is proposed, has a molecular weight of 57,000. Antibodies against vimentin and antibodies against prekeratin have been used in parallel in immunofluorescence microscopy on a variety of cultured cells as well as on frozen tissue sections. Both antibodies decorate extended wavy arrays of filaments that are different from microfilaments and microtubules. Intermediate filament bundles decorated by antibodies against prekeratin are predominant in many epithelial cells, including epithelia-derived tumor cells, and are not decorated by antibodies to vimentin. In contrast, intermediate filaments decorated by antibodies against vimentin are widespread among nonmuscle cells of mesenchymal origin, including transformed cells, and also occur in other cells. Perinuclear whorls of aggregates of intermediate filaments induced by prolonged treatment with Colcemid generally show strong decoration with antibodies against vimentin. No significant reaction with either antiserum has been observed in muscle structures or in brain nerve tissue. These observations show that intermediate filaments with similar ultrastructure and solubility characteristics can be distinguished immunologically.