We found that, compared to the perilesional or control normal fibroblasts, all cultures derived from cholesteatoma samples were less proliferating and more differentiated; moreover, although very variable among the cultures, the differentiated phenotype well correlated with an activated state, shown by MMP-2 and MMP-9 secretion, demonstrating that also in a non-neoplastic context, such as the cholesteatoma disease, the associated fibroblasts might exert a pathological role through biological functions which are maintained in culture. Here, MMP2 is linked to cholesteatoma.