Breast Tumor Kinase (Brk/PTK6) has a relatively limited expression profile in normal tissue. Its expression is restricted to epithelial cells that are differentiating such as those in the epidermis, and Brk expression appears to be absent from proliferating cells in normal tissue. Also, there is now some evidence to suggest that Brk plays a functional role in the differentiation of the keratinocytes in the epidermis. We have, therefore, investigated the role that Brk/PTK6 plays in normal human primary keratinocytes by suppressing protein levels using RNA interference. We show that as primary human keratinocytes are induced to differentiate in vitro, Brk levels decrease. Decreasing Brk protein levels lead to an increase in the number of cells with a permeable plasma membrane, a decrease in epidermal growth factor receptor (EGFR) and a parallel increase in keratin 10 levels, but classical markers of apoptosis or terminal differentiation are not affected. We propose Brk, Keratin 10 and EGFR are co-regulated during differentiation and that manipulating Brk expression can influence the differentiation of normal primary human keratinocytes.