Mouse monoclonal antibodies (MAb) were generated against p33ING1b tumor suppressor protein. 15B9 MAb was highly specific in recognizing a single protein band of ∼33 kDa endogenous p33ING1b protein from HCC cell lines and normal liver tissue by Western blot analysis and by immunoprecipitation. Although p33ING1b mutations are rarely observed in cancer, differential subcellular distribution and nuclear exclusion of p33ING1b were reported in different cancer types. Therefore we analyzed the expression and subcellular localization of p33ING1b in HCC cell lines using 15B9 MAb. So far, p33ING1b mutations or differential subcellular localization are not reported in HCC. In this study, by indirect immunofluorescence using MAb 15B9, we demonstrate that nuclear localization of p33ING1b was highly correlated with well-differentiated HCC cell lines whereas poorly differentiated HCC cells have nuclear exclusion of the protein. Moreover no association was observed between differential subcellular localization of p33ING1b and p53 mutation status of HCC cell lines. Hence our newly produced MAb 15B9 can be used for studying cellular activities of p33ING1b under normal and cancerous conditions. © Copyright 2009, Mary Ann Liebert, Inc.