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CD63 is a member of the tetraspanin superfamily of membrane glycoproteins that has been hypothesised to provide a structural network in the organisation of large multimolecular microdomains at cell membranes. Detailed analyses of the role of CD63 in these complexes through mutagenic studies have been limited, however, by the ubiquitous cellular expression of CD63 in vivo and in vitro. In an attempt to define CD63-null cell lines, we have analysed the expression of CD63 and other tetraspanins on a panel of human cancer cell lines. Similar expression patterns were seen between cell lines from melanomas, breast cancers and prostate cancers. The melanoma cell line KM3, however, described previously as a CD63-null human cell line, was found to express none of the 7 human tetraspanins tested. KM3 was identified definitively as a rat cell line by analysis of karyotype and antigen expression. Notably, KM3 was found to express the rat homologue of CD63. Conclusions concerning the function of human CD63 drawn from studies using KM3 cells therefore require re-evaluation as does the frequently cited hypothesis that CD63 expression is linked to melanoma progression. As KM3 is the only cell line thus far identified as CD63 negative, these results highlight the necessity for the production of a CD63 null system.