From collagenase digests of human thyroid, endothelial cells were separated from follicular cells by their greater adherence to gelatin-coated plates. Endothelial cells were further purified using fluorescence-activated cell sorting, selecting for cells expressing factor VIII-related antigen. Isolated cells were negative for thyroglobulin and calcitonin when examined by immunostaining. The receptor for the angiopoietins, Tie-2, was expressed by the cells and expression was increased by agents that elevate cAMP. Nitric oxide synthase 3 (NOS-3), the endothelial form NOS was expressed by the cells and similarly regulated. Cells responded strongly to the mitogen FGF-2 in growth assays but only weakly to vascular endothelial growth factor (VEGF). VEGF was however able to stimulate NO release from the cells consistent with their endothelial origin. The FGF receptor (FGFR1) was full length (120kDa) and immunolocalized to the cytosol and nucleus. TSH did not regulate FGFR1 but its expression was increased by VEGF. Thrombospondin, a product of follicular cells, was a growth inhibitor but neither TSH nor T₃ had direct mitogenic effects. Thyroid follicular cell conditioned medium contained plasminogen activator activity and stimulated the growth of the endothelial cells but when treated with plasminogen to produce the endothelial-specific inhibitor, angiostatin, growth was inhibited. Human thyroid endothelial cell cultures will be invaluable in determining the cross talk between endothelial and follicular cells during goitrogenesis.