Detailed characterization and identification of cancer cell lines is the basis for the credibility of experimental studies. Therefore, chromosomal analysis should be routinely included in the protocol of cell line characterization and in the protocols of experimental studies performed on cell lines. In 2000, our group established and characterized cytomorphologically and immunophenotypically a new cell line, OvBH-1, which was derived from the ascitic fluid cells of an untreated patient with ovarian clear-cell adenocarcinoma. The aim of the current study was to characterize OvBH-1 cytogenetically and to monitor its stability by comparison of morphologic, immunohistochemical, and cytogenetic features between the early (135) and late (385) passages. Conventional and molecular cytogenetic analyses (fluorescence in situ hybridization and spectral karyotyping) of OvBH-1 revealed the following hypotriploid karyotype with random translocations: der(2)t(2;13),der(4)t(4;22), der(5)t(2;5). Complex rearrangements involving chromosomes 3, 15, and 20 were also found. FISH analysis with a p53 probe indicated the deletion of this region in two out of three copies of chromosome 17. The morphologic and immunophenotypic features, as well as the karyotypes observed in OvBH-1 in passages 135 and 385, were comparable. The monoclonality of the cell line was confirmed in a single cell cloning experiment. Our study indicated that OvBH-1 is characterized by a distinct karyotype and remains stable over 250 passages. Taking into account its thermosensitivity, its unusual karyotype, and its stability, this line can be considered as a valuable model for various experimental studies.