Oral squamous cell carcinoma (OSCC) develops along a multistep genetic pathway including loss of tumor suppressor genes and alteration of oncogenes. We characterized seven OSCC cell lines by classical and molecular cytogenetic analysis and fresh tumor and adjacent oral mucosa corresponding t o three of the cell lines by molecular cytogenetics. We observed homogeneously staining regions (hsrs) in four of the seven cell lines, at I I q I3 in three and at I lq23 and in an unidentified marker chromosome in the fourth. Amplification of band I I q I 3 occurs in 30-6076 of head and neck squamous cell carcinomas. To determine whether INJ2 and HSTI, both located in band I I q 13, are amplified in the tissues and cell lines and t o confirm the chromosomal location(s) of the amplification, we used dual-color fluorescence in situ hybridization (FISH) with DNA probes for these genes and the chromosome I I centromere. We report chromosomal localization of INTZIHSJI amplification in OSCC. Coamplification of INT2 and HSTI was detected in the hsrs in cultured tumor cells from the four hsr-containing tumors and in directly harvested tumor cells, which were available from only two of these tumors. Amplification was not present in tumors lacking hsrs or adjacent oral mucosa corresponding t o any of the seven tumors. The observation of amplification in fresh tumor cells suggests that the amplification was present in the patients, may play a key role in the development andlor progression of OSCC, and is not due t o karyotypic evolution in vitro. The absence of amplification in the adjacent mucosa suggests that I I q I 3 amplification is a relatively late event in OSCC tumorigenesis. Genes Chromosom Concer 12: 288-295 (1995).