Ascitic cytological diagnosis is critical, but ovarian adenocarcinoma cells and reactive mesothelial cells can be difficult to distinguish because they usually have atypical cell nuclei and increased nuclear/cytoplasmic ratios. Previous studies using DNA microarrays have demonstrated that hepatocyte nuclear factor-1beta (HNF-1beta) is expressed specifically in clear cell adenocarcinoma (CCC). Thus, in the present study, we investigated the usefulness of HNF-1beta as an immunocytochemical diagnostic marker of CCC in ascitic specimens. We first confirmed that HNF-1beta expression levels were significantly higher in CCC than in non-CCC (i.e. serous adenocarcinoma, mucinous adenocarcinoma and endometrioid adenocarcinoma) in 55 surgical specimens at both the mRNA (P < 0.05) and protein (P < 0.05) levels by real-time polymerase chain reaction and immunohistochemistry, respectively. Immunocytochemistry of 60 cytological specimens showed significant positivity in CCC cases whereas all non-CCC cells, except for three endometrioid adenocaricnoma cases, and mesothelial cells in the background stained negatively for anti-HNF-1beta antibody (P < 0.05). The sensitivity and specificity were calculated to be 0.955 and 0.921, respectively. Immmunostaining patterns of HNF-1beta on cytological specimens were similar to those observed on histopathological ovarian specimens from the same patients. Double immunohistochemical staining using anti-HNF-1beta antibody and HBME-1, a mesothelium-specific monoclonal antibody, confirmed that anti-HNF-1beta antibody distinguished CCC cells and mesothelial cells. In conclusion, our findings indicate the specific expression of HNF-1beta in ovarian CCC and possible clinical applications of HNF-1beta immunocytochemical staining for the differential cytopathological diagnosis of CCC from non-CCC, as well as from mesothelial cells using cytological specimens from ovarian carcinoma patients.