BACKGROUND AND METHODS: In Paget's disease of the breast, the epidermis of the nipple is infiltrated by large neoplastic cells of glandular origin. It has been hypothesized that the spread of Paget cells through the nipple epidermis is induced by a motility factor that acts via the HER2/NEU receptor. To test this hypothesis, we characterized and purified a motility factor released by keratinocytes and identified its target receptors in specimens from patients with Paget's disease and in SK-BR-3 breast adenocarcinoma cells, which overexpress HER2/NEU. RESULTS: We isolated the motility factor from keratinocyte-conditioned medium and sequenced tryptic peptides. These sequences were used to identify the motility factor as heregulin-alpha, which is released by skin keratinocytes. Heregulin-alpha induces spreading, motility, and chemotaxis of SK-BR-3 cells, as does motility factor. Motility factor activities of heregulin-alpha are inhibited by monoclonal antibody AB2, directed against the extracellular domain of HER2/NEU, which blocks the binding of heregulin-alpha. We used in situ hybridization to show that normal epidermal cells produce heregulin-alpha messenger RNA and that heregulin receptors, HER3 and/or HER4, as well as their coreceptor HER2/NEU, are expressed by Paget cells. CONCLUSIONS: Heregulin-alpha is a motility factor that is produced and released by normal epidermal keratinocytes and thus plays a key role in the pathogenesis of Paget's disease. Paget cells express heregulin receptors HER2/NEU, as well as HER3 and/or HER4, both of which function as a co-receptor of HER2/NEU. Binding of heregulin-alpha to the receptor complex on Paget cells results in the chemotaxis of these breast cancer cells, which eventually migrate into the overlying nipple epidermis.