Head and neck cancer (HNC) patients are at high risk of developing second primary tumors of the upper aerodigestive tract and this is a chief cause of death. Genomic instability reflecting the propensity and the susceptibility of the genome to acquire multiple alterations is considered a driving force behind multiple carcinogenesis and the alterations of the length of single repetitive genomic sequences or microsatellite instability (MI), implicating impaired DNA repair mechanisms, and could be a sensitive marker for assessing genomic instability in multiple HNC. To investigate whether a genetic defect(s) involving the mismatch repair system constitutes a risk factor in patients with multiple head and neck cancer, we examined replication errors (RER) at 10 microsatellite loci in 21 primary and 5 second primary cancers in 21 patients with multiple malignancies of the upper aerodigestive tract, in comparison with match-paired primary HNC from patients without multiple malignancies. A RER+ phenotype (alterations at > or =2 loci) was observed at 10 microsatellite alterations on chromosomes 2, 3, 11, 17 in at least one tumor from 15 out of 21 (71.5%) patients with multiple primary cancers but only in 11 tumors from 40 (27.5%) HNC patients with single cancer (P=0.001). A RER+ phenotype was also associated with a positive familial cancer history (P=0.046). Our results suggest that a genetic instability may play an important role in the pathogenesis of multiple primary cancers and that testing for MI in a primary HNC may be useful in detecting patients with high risk for developing multiple malignancies of the upper aerodigestive tract.