Thyroid cancer sometimes contains poorly differentiated components, which have the potential of invasion and metastasis. We evaluated the possible roles of heparin-binding EGF-like growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family, in cell growth and invasion of thyroid cancer cells, and demonstrated that HB-EGF is not only a potent mitogen but also a chemotactic factor in the thyroid cancer cells 8305C and SW579. The HB-EGF-mediated chemotaxis was inhibited by neutralizing antibody against the EGF receptor (EGFR/HER1/ErbB1) or tyrphostin AG1478, a specific inhibitor of the EGFR tyrosine kinase. The HB-EGF mRNA and protein expression was also analyzed using RT-PCR and immunofluorescence methods, respectively. In addition, in clinical immunohistochemical study, increased expression of HB-EGF and its receptors, HER1 and EGFR4 (HER4/ErbB4), was observed in thyroid carcinoma cells. Our findings suggest that HB-EGF acts as a potent paracrine and/or autocrine chemotactic factor as well as a mitogen that mediates HER1 and/or HER4 in the invasion and metastasis of thyroid carcinoma cells, including poorly differentiated papillary carcinomas or undifferentiated/anaplastic carcinomas. These data may aid in the development of novel therapeutic strategies for thyroid cancer.