Serine proteases cause aggregation of the rat ascites tumor cell lines AH-130, AH-109A and YS in vitro, and the tumor cell aggregates are dissolved by treatment with DNase I. We previously demonstrated that these events played a critical role in the augmentation or reduction of experimental blood-borne metastasis of these cell lines. In the present study, the ultrastructural features of this protease-dependent aggregation were analysed. Transmission and scanning electron microscopy revealed that after the protease treatment each tumor cell was surrounded by a thin membranous (sleeve-like) structure. This sleeve-like structure was stained with ruthenium red to an intensity similar to the cell surface of the control. Adjacent cells became attached to each other with microvilli via this fine structure. Immuno-electron microscopy revealed DNA antigen as dense patches on the sleeve-like structure or as faint and diffuse deposits on the outer surface of the cells by indirect immunoperoxidase staining using an anti-DNA monoclonal antibody. Both the sleeve-like structure and immunopositive deposits disappeared after treatment with DNase I. Neither cell viability nor the normal ultrastructure of their organelles was influenced by the enzyme treatment. These results indicate that serine protease-induced tumor cell aggregation is due to cellular contact via the sleeve-like structure, which probably originates from the cell surface glycocalyx in association with DNA molecules of unknown origin.