Homogeneous staining region (hsr), a cytogenetic indicator of gene amplification, has been frequently found in ovarian carcinoma (ovc). To identify the origin of the hsr, chromosome microdissection combined with polymerase chain reaction and fluorescence in situ hybridization (FISH) was applied to two human ovarian cancer cell lines, GR and MLS/P. The hsr probes were labeled with biotin or digoxigenin and hybridized to normal metaphase spreads to elucidate the chromosomal origin and regional localization of the amplified genes. FISH to normal metaphase spreads with the probe generated from the whole hsr-bearing chromosome from GR hybridized to 8q24, 2p13 →2q11.2, 10pter →10p15, 10p12 →10q11.2, 5q23 →5q31, and 5q33 →5qter. For MLS/P, the hsr-bearing marker chromosome hybridized to 8q and 15q. In both cases, detailed FISH analysis revealed enhanced signal intensity at the 8q24 locus, which coincides with the chromosomal location of the C- MYC oncogene. To verify the involvement of C- MYC in hsr formation, in situ hybridization with a probe specific for the C- MYC oncogene was conducted and confirmed the amplification of C- MYC as the origin of the hsr. The whole hsr-bearing chromosome for GR is designated as rev ish der(10) (10pter →10p15::8q24hsr:: 10p12 →10q11.2::8q24::2q11.2 →2p13::2p13 →2q11.2::8q24::10q11 →10p11.2::5q23 →5q31::5q33 →5qter (wcp10 +,D10Z1 ++,wcp2 +,D2Z ++,wcp5 +,wcp8 +,C-MYC ++/hsr). The hsr-bearing marker for MLS/P is designated as rev ish der(8)(qter →8q24::8q24::8q24 →8q10::8q10 →8q24::8q24::8q24::8q24 →8qter:: 15q11 →15qter)(wcp8 +,D8Z1 +,wcp15 +,C- MYC+++). FISH with the probe generated from the hsr of GR also painted the hsr in MLS/P, indicating that the two hsrs have shared homology, which indicates that the amplification of 8q24/C- MYC as the origin of hsr may be a nonrandom genomic alteration in ovc.