Thyroglobulin is the major secretory protein of thyroid epithelial cells. Part of thyroglobulin reaches the circulation of vertebrates by transcytosis across the epithelial wall of thyroid follicles. Clearance of thyroglobulin from the circulation occurs within the liver via internalization of thyroglobulin by macrophages. Here we have analyzed the interaction of thyroglobulin with the cell surface of J774 macrophages with the aim to identify the possible thyroglobulin-binding sites on macrophages. Binding of thyroglobulin to J774 cells was saturated at approximately 100 nM thyroglobulin with a Kd of 50 nM, and it was competed by the ligand itself. Preincubation of J774 cells with thyroglobulin resulted in downregulation of thyroglobulin-binding sites, indicating internalization of thyroglobulin and its binding proteins. By affinity chromatography, two proteins from J774 cells were identified as thyroglobulin-binding proteins with an apparent molecular mass of approximately 33 kD. Unexpectedly, both proteins were identified as histone H1 by protein sequencing. The occurrence of histone H1 at the plasma membrane was further proven by biotinylation or immunolabeling of J774 cells. The in vitro interaction between histone H1 and thyroglobulin was analyzed by surface plasmon resonance that revealed a Kd at 46 nM. In situ, histone H1 was colocalized to FITC-Tg-containing endocytic compartments of Kupffer cells, i.e., liver macrophages. We conclude that histone H1 is detectable at the cell surface of macrophages where it serves as a thyroglobulin-binding protein and mediates thyroglobulin endocytosis.