Antibody 3-40 defines a 35- to 40-kd surface antigen present on T lymphoblastic leukemia (T-ALL) cells that is absent on normal hematopoietic cells (Naito et al, Blood 62:852, 1983). Using immunoblot analysis of cytoskeletal proteins and indirect immunofluorescence of cell lines treated with various cytoskeletal inhibitors, in this report we have demonstrated that antibody 3-40 also identifies vimentin intermediate filaments (IMF) within the HSB-2 (T-ALL) cell line as well as in normal thymocytes, peripheral blood mononuclear cells, and human and rodent fibroblast cell lines. Cross-reactivity with several keratin subclasses was demonstrated in both human and rodent epithelial cell lines, human thymus, and skin. In addition, we have shown that antibody 3-40 defined a 39-kd intracellular IMF-associated protein in HSB-2 cells, epithelial and fibroblast cell lines. This IMF-associated protein may be selectively expressed on the surface of human T cells during malignant transformation.