Background: Squamous cell carcinoma of the head and neck (SCCHN) is one of the 10 most frequently occurring cancers in the world. Defective mismatch repair, as exhibited by the phenomenon of microsatellite instability, has been observed in SCCHN although no reports of mismatch repair gene mutations or altered protein expression have been published. In a variety of microsatellite instability (MSI) positive cancers where mutations in the mismatch repair (MMR) genes were not observed, allelic imbalance at the loci of the MMR genes was prevalent. Objective: To investigate whether allelic imbalance at the MMR genetic loci contributes to the development of SCCHN. Materials and Methods: 35 matched normal/tumour SCCHN pairs were studied using 29 microsatellite markers located within and adjacent to six known DNA mismatch repair genes. In addition, mutational analysis and protein expression of hMSH2 and hMLH1 were investigated. Results and conclusions: We demonstrated that 36 and 17% of the analysed SCCHN specimens exhibited allele imbalance at the hMLH1 and hMSH3 genetic loci, respectively. Allelic instability at these two loci was found to be correlated with the MSI status of the SCCHN tumours. Allelic instability was found to be uncommon at the other MMR gene loci analysed. One mutation was found in hMSH2 and none in hMLH1 in this series of tumours. 23 of 24 (96%) of the examined SCCHN tumours showed reduced expression of either hMSH2 or hMCH1 genes. Allelic instability in the MMR genes, hMLH1 and hMSH3, is proposed to be involved in the aetiology of SCCHN tumours.