Monoclonal antibody (mAb) U36 was developed for the treatment of minimal residual disease of head and neck squamous cell carcinoma (HNSCC). The mAb- U36-defined antigen was characterized by cDNA cloning, and was shown to be identical to the keratinocyte-specific CD44 splice variant epican. The epitope recognized by mAb U36 was shown to be located in the v6 domain. Two amino acids within the epitope appeared to differ from the sequences that have been described in literature. The sequence of the epitope appeared to contain glutamic acid at position 367 and lysine at position 374, while valine and arginine respectively have been described before. Interestingly, another anti-CD44v6 antibody with possible clinical application, VFF18, recognizes an epitope in the same area. With respect to the applicability of these antibodies for tumor targeting, this variation might have an influence on antibody-antigen interaction and mAb accumulation in the tumor. Furthermore, this observation raised the question whether the different epitopes are related to the malignant behavior of tumor cells. In this paper we determine the relative affinity of mAb U36 for the variant epitope sequences by tumor cell binding assays using synthetic peptides for competition. The presence of glutamic acid instead of valine at position 367 caused strong competition. Further evaluation showed that the published valine variant does not exist in vivo, and is the result of a sequencing artefact. The effect of substitution of lysine for arginine at position 374 had no effect on the binding of mAb U36 to the cells. This amino acid variation was shown to be due to allelic polymorphism. There was no trend towards allelic imbalance in tumor cells as compared to normal cells.