Concurrent DNA flow cytometric (FCM) and fluorescence in situ hybridization (FISH) analyses were prospectively performed on 24 primary untreated head and neck squamous carcinomas for characterization of the genotypic and phenotypic DNA aberrations of these neoplasms. Eleven tumors (42.0%) manifested DNA diploidy (DI = 1.00) and 15 (58.0%) had DNA aneuploidy (DI < or > 1.00) by FCM. Fluorescence in situ hybridization results showed aneusomy in the majority of DNA diploid and in all DNA aneuploid tumors. The extent of the abnormalities for individual chromosomes and the number of involved chromosomes in a given DNA diploid or aneuploid tumor were significantly different. Overall, a statistical correlation between the FCM DNA index (DI) and the magnitude of the chromosomal aberration by FISH was found. Our results also show a significant association between the DI and histologic differentiation and stage of disease in these neoplasms. In conclusion, (1) chromosomal aneusomy characterizes most DNA diploid (DI = 1.00) and all DNA aneuploid (DI < or > 1.00) head and neck squamous carcinomas; (2) polysomy is the most prevalent finding; (3) loss of the Y chromosome in tumors from male patients is a consistent feature; (4) the FCM DI reflects net chromosomal gains or losses in these neoplasms; and (5) DNA aneuploidy is associated with tumor aggressiveness.