Mitochondrial DNA (mtDNA) mutations in coding and noncoding regions have been reported in a variety of human cancers. Despite a greater number of studies, the relationship between such alterations and nuclear microsatellite instability (nMSI) of the tumor cells remains controversial. To contribute new data to this discussion, we investigated head and neck squamous cell carcinomas (HNSCC) for mutations and mitochondrial microsatellite instability (mtMSI) in 2 parts of the mitochondrial D-loop as well as mutations in 2 mitochondrial genes and for the delta4977 mtDNA deletion. These results were compared with data of an analysis for microsatellite instability at IGFIIR, hMSH3, hMSH6, and 5 dinucleotide repeats. We found mtMSI, low nMSI, and high nMSI in 42%, 36%, and 13% of HNSCC primary tumors, respectively. A de novo delta4977 mtDNA deletion could be demonstrated in 25% of HNSCCs. A correlation between mtMSI and nMSI or between a de novo occurrence of the delta4977 mtDNA deletion and nMSI could not be detected in our HNSCC samples (P values 0.527 and 0.078, respectively). Nevertheless, the high rate of mtMSI suggests an involvement of mtDNA alterations in the tumorigenesis of this head and neck cancer and supports the proposal that this aberration may be a new tumor marker.