The current paucity of cytogenetic information on hepatocellular carcinoma (HCC) reflects the difficulties in culturing hepatocytes in vitro. Here, we report on the successful culture of 15 HCC cases. Chromosome aneuploidy ranging from a near-diploid to hyperhexaploid karyotype was found, but their complete karyotypic interpretations were hampered by the presence of many unidentifiable rearrangements. Spectral karyotyping (SKY) was used to elucidate structural changes in these HCC samples and 3 liver cancer cell lines (PLC/PRF/5, Hep3B, and HepG2). Frequent structural abnormalities were found on chromosomes 1 (13 of 15 cases; 3 of 3 cell lines), 8 (10 of 15 cases; 2 of 3 cell lines), 17 (9 of 15 cases; 3 of 3 cell lines), and 19 (9 of 15 cases; 1 of 3 cell lines). In particular, the chromosome regions 1p13-q21, 8p12-q21, 17p11-q12, 17q22, and 19p10-q13.1 were involved in multiple rearrangements. SKY analysis also suggested several previously undescribed breakpoints in HCC. These breakpoints, predominantly pericentromeric, clustered around the chromosome bands 2q33-q34, 3p13-q12, 4p14-q12, 5p10-q11, 7p12-q11, 10q10-q11, 11q10, 11q13-q21, 12q10-q13, 12q22-q23, 13q10-q14, 15q10, 16q10-q13, 18p11-q11, 20p11-q13.1, 21q10, and 22q10. When tumor sizes were compared, a significantly higher number of structural abnormalities was found in tumors larger than 4 cm (P = .007). Rearrangements such as t(1;8), t(1;11), t(1;19), and t(17;21) that were identified in both primary tumors and cell lines might represent markers that reflect proliferative advantages. Although SKY analysis did not indicate consistent translocations, it suggested nonrandom breakpoints, predominantly in the pericentromeric region, on a number of chromosomes. These breakpoint clusters may thus prove to be more important in the liver carcinogenesis and targets for further molecular investigations.