Hypothesis: Cultured fibroblasts derived from experimental gerbil cholesteatoma tissue exhibit an invasive phenotype in comparison with normal fibroblasts. Background: Aural cholesteatomas are enlarging accumulations of keratin debris caused by keratinizing squamous epithelium in the middle ear. They characteristically result in the destruction of adjacent tissues, specifically bone erosion. The mechanisms by which cholesteatomas relentlessly invade the structures of the temporal bone are varied, but it has been suggested that one factor contributing to the aggressive nature of cholesteatomas is the transformation of resident fibroblasts into an invasive phenotype. Methods: The ability of cultured normal and cholesteatoma fibroblasts to invade a basement membrane matrix in a Boyden chamber assay was examined. Results: Less than 1% of gerbil fibroblasts invaded the matrix, compared with almost 10% of the invasive HT-1080 fibrosarcoma cells. Normal and cholesteatoma fibroblasts did not differ from each other in their invasive potential. Conclusion: Normal fibroblasts and fibroblasts from induced cholesteatomas do not exhibit the invasive phenotype characteristic of true neoplastic cells.