RBP-Jx is a novel type of transcriptional regulatory protein that does not contain any known DNA-binding motif. We raised anti-RBP-Jx monoclonal antibodies (K0043 and T6709) to investigate the roles of RBP-Jx in cell differentiation. These antibodies stained nuclei of undifferentiated embryonic stem cells and F9 cells but not those of the other differentiated cell lines tested so far although the RBP-Jx protein is expressed at similar levels. Interestingly, differentiated F9 cells lost the immunostaining reaction with the antiRBP-Jx monoclonal antibodies. Biochemical subcellular fractionation study showed that the majority of RBP-Jx was localized in nuclei of F9 cells and that there are at least two forms of the RBP-Jx protein in the nuclei of undifferentiated F9 cells, a free form and a chromatin-bound form. Upon induction of F9 cell differentiation, free nuclear RBP-Jx disappeared concomitantly with the loss of immunostaining, suggesting that the anti-RBP-Jx antibodies cannot recognize chromatin-bound RBP-Jx. Since there is no evidence to indicate covalent modification of RBP-Jx, we assume that chromatin-bound RBP-Jx interacts with a large number of proteins which block the exposure of RBP-Jx epitopes to the monoclonal antibodies.