In order to investigate the subnuclear interactions of the WT1 gene product, nuclear fractionation analyses were performed with human osteosarcoma HOS and myelogenous leukemia K562 cells. The WT1 protein was tightly associated with the nucleus and was resistant to high-salt or detergent extraction and DNase I digestion. Both the expression level and stability of WT1 and its resistance to high salt and DNase I treatments remained constant during the cell cycle. In addition, human WT1 ectopically expressed in mouse NIH3T3 cells was also resistant to these treatments. These results suggest that WT1 functions in tight association with the nuclear matrix.