Endothelin (ET) receptor dysregulation has been described in a number of pathophysiological processes, including cardiovascular disorders, renal failure, and cancer. The aim of this study was to evaluate the expression of the ET-A receptor (ET(A)R) in murine models of thyroid carcinoma using optical imaging methods. A recently developed near-infrared fluorescent tracer was first assessed in isolated artery preparations for its functional performance in comparison with known ET(A)R antagonists BQ123 and PD156707. Before evaluation of the tracer in vivo, different thyroid carcinoma cell lines were characterized with respect to their ET receptor expression by RT-PCR and autoradiography. In vivo, sc and orthotopic papillary thyroid tumor xenografts were clearly visualized by fluorescence reflectance imaging and fluorescence-mediated tomography up to 48 h after injection of the tracer. Binding specificity of the probe was demonstrated by predosing with PD156707 as a competing inhibitor. In conclusion, optical imaging with a fluorescent ET(A)R tracer allows the noninvasive imaging of tumor-associated ET(A)R expression in vivo. In the future, this technique may help surgeons to evaluate lesion dimensions in intraoperative settings (e.g. thyroidectomy).