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.