The Pebpb2/Cbfb gene encodes the non-DNA binding subunit of the heterodimeric transcription factor, PEBP2/CBF. To examine the expression of the PEBP2beta/CBFbeta protein in vivo, we carried out immunohistochemistry using the tissues from adult mice as well as embryos. Although PEBP2beta/CBFbeta was detected in various tissues to various degrees, interesting features of expression were observed in the skeletal myogenic cells. Here PEBP2beta/CBFbeta was found mainly to occur as cytoplasmic staining and the intensity of this staining increased depending on the differentiation stage of the cells. In the undifferentiated myoblasts PEBP2beta/CBFbeta was undetectable, whereas moderate levels of PEBP2beta/ CBFbeta were detected in the elongated and aligned myocytes. PEBP2beta/CBFbeta appeared to accumulate further when the cells fused to each other to become multinucleated myotubes. Once the muscle fibers were established, PEBP2beta/CBFbeta was relocated onto or around the Z-lines. PEBP2beta/CBFbeta was also detected in the cytoplasm of cardiac myocytes and in the smooth muscle cells of the digestive tract. In all the above, the skeletal myotubes were the only case that showed both nuclear and cytoplasmic staining of PEBP2beta/CBFbeta. Thus, we could show differentiation dependent pattern of PEBP2beta/CBFbeta expression in muscle development and establish PEBP2beta/CBFbeta to be a cytoplasmic as well as nuclear protein in vivo.