The CCAAT/enhancer-binding protein (C/EBP)β belongs to the C/EBP family of proteins that possesses a basic leucine zipper DNA-binding domain. These proteins bind DNA by dimerization and play a role in the transcriptional regulation of various cells. There are six different types of C/EBPs, and some form isoforms through the use of alternative translation initiation sites. The functional analysis of the C/EBP family is therefore difficult to achieve. Here we report on the production of specific monoclonal antibodies against mouse C/EBPβ using a rat medial iliac lymph node method. Immunoblotting using C/EBPβ monoclonal antibodies identified two types of isoforms, while immunostaining revealed a subnuclear localization for C/EBPβ. Use of this antibody should contribute to the further elucidation of the transcriptional regulatory function of C/EBPβ.