The Pebpb2/Cbfb gene encodes the non-DNA binding beta subunit of the heterodimeric transcription factor, PEBP2/CBF, and has been implicated in a subtype of human acute myeloid leukemia, as well as being indispensable for the development of definitive hematopoiesis in the murine fetal liver. By examining a subcellular localization of the PEBP2beta/CBFbeta protein in tissue culture cells, we could reveal an additional aspect of the protein other than to be a subunit of a transcription factor. Immunoblot and immunocytochemical staining showed that PEBP2beta/CBFbeta was mostly present in the cytoplasm. This PEBP2beta/CBFbeta was free from its DNA-binding partner, the alpha subunit of PEBP2/CBF, as judged by the electrophoretic mobility shift assays. Furthermore, a significant amount of PEBP2beta/CBFbeta was retained in the cytoskeleton preparation after detergent extraction of the cells and was found by double immunofluorescence to colocalize with the F-actin on stress fibers and the vinculin in membrane processes. Thus, the present study extends PEBP2beta/CBFbeta to be a cytoskeleton-affinitive as well as nuclear protein. The implications of these results are discussed.