Comparative Genomic Hybridization (CGH) is a novel cytogenetic method that allows the comprehensive analysis of a tumor genome for DNA gains and losses. CGH was performed on genomic DNA extracted from 14 primary head and neck squamous cell carcinomas. Equal amounts of biotin-labeled tumor DNA and digoxigenin-labeled normal reference DNA were hybridized to normal metaphase chromosomes. The tumor DNA was visualized with fluorescein (FITC) and the normal DNA with rhodamine (TRITC) and detected in a fluorescence microscope. The signal intensities of the different fluorochromes were quantitated as gray levels along the single chromosomes. The over-and underrepresented DNA segments were quantified by computation of FITC/TRITC ratio images and average ratio profiles. Consensus deletion regions were most frequently observed on chromosome arms 3p (14 cases), 9p (11), 13q (10), 18q (10), 5q (9), 4q (9), 4p (7), 11q (7), 6q (6), 8p (6), and 11p (6). Copy number increases were identified for chromosomes 3q (11), 16p (9), 17q (9), 19p (8), 19q (7), 22 (7), 1p (6), 8q (6), 9q (6), and 20q (6). Particularly, the 3q isochromosome formation (3p loss/3q amplification), found in 10 cases, was a basic alteration. In addition, 11 tumors showed a 11 q13 amplification. CGH analysis allows the identification of recurrent genetic alterations in head and neck squamous cell carcinomas that will be associated with specific tumor phenotypes like metastatic behavior and prognosis.