Biopsies of oropharyngeal cancer were screened for chromosomal imbalances by comparative genomic hybridization (CGH) performed on 22 primary tumors and morphologically nonmalignant surrounding mucosa. The aim was to determine early chromosomal changes of tumor development and to draw conclusions on the mechanisms leading to multiple tumors. The most prominent chromosomal imbalances observed were over representations of genomic material on 3q, 15q, 8q, and 11q and losses on 9p, 3p, and 11q. In morphologically normal mucosa collected at 1 cm from the primary tumor border (M1), amplifications on 15q and 21q were most frequent. Far fewer gains and losses were found in M1 than in the primary tumor (average 2.2 vs. 6.9). Gains dominated over losses, but a tendency toward an increasing proportion of losses in the primary tumor (PT) was observed (ratio of gains to losses: PT, 4.75; M1, 6.3). Almost all the imbalances in M1 were detected in the primary tumor. No chromosomal alterations were identified with CGH in tissue samples dissected at 2 cm from the primary tumor (M2). In all samples, dysplastic morphologic changes decreased with distance from the primary tumor, which correlates with the observed lower level of genetic changes. We suggest that gains of genetic material on 15q and 21q are early events in malignant progression of squamous cell carcinoma, followed by gains on 3q, 8q, and 11q, and losses on 3p and 9p at later stages. Based on our cytogenetic data, we discuss the monoclonal model followed by lateral epithelial spread as an explanation of multiple head and neck squamous cell carcinomas.