Radixin is a barbed end-capping actin-modulating protein which was first identified in isolated cell-to-cell adherens junctions from rat liver (Tsukita, Sa., Y. Hieda, and Sh. Tsukita, 1989. J. Cell Biol. 108:2369-2382). In the present study, we have analyzed the distribution of radixin in dividing cells. For this purpose, an mAb specific for radixin was obtained using chicken gizzard radixin as an antigen. By immunofluorescence microscopy with this mAb and a polyclonal antibody obtained previously, it was clearly shown in rat fibroblastic cells (3Y1 cells) that radixin was highly concentrated at the cleavage furrow during cytokinesis. Radixin appeared to accumulate rapidly at the cleavage furrow at the onset of furrowing, continued to be concentrated at the furrow during anaphase and telophase, and was finally enriched at the midbody. This concentration of radixin at the cleavage furrow was detected in all other cultured cells we examined: bovine epithelial cells (MDBK cells), mouse myeloma cells (P3 cells), rat kangaroo Ptk2 cells, mouse teratocarcinoma cells, and chicken fibroblasts. Furthermore, it became clear that the epitope for the mAb was immunofluorescently masked in the cell-to-cell adherens junctions. Together, these results lead us to conclude that radixin is present in the undercoat of the cell-to-cell adherens junctions and that of the cleavage furrow, although their respective molecular architectures are distinct. The possible roles of radixin at the cleavage furrow are discussed with special reference to the molecular mechanism of the actin filament-plasma membrane interaction at the furrow.