Osteopontin (OPN) is a secreted acidic, phosphoglycoprotein with a central GRGDS integrin binding domain. We have previously shown that OPN mediates chemotactic migration of Langerhans-/Dendritic Cells (LC/DC) to lymphnodes, thereby playing a central role during the sensitization phase of allergic contact hypersensitivity. Thrombin cleavage of OPN, close to the RGD sequence, results in two fragments of similar size but with distinct biological functions. The N-terminal fragment contains the av binding RGD sequence, while the C-terminal fragment contains the predicted CD44 binding domain. OPN has a modular structure with multiple functional domains and its biological activity is influenced by its state of glycosylation and phosphorylation. In vivo, OPN fragments cleaved at inflammatory sites may influence DC-activation and migration. Here we investigated the secondary structure of recombinant eucaryotic OPN and compared its biological activity with thrombin cleaved fragments, neuraminidase treated OPN and a procaryotic OPN-GST-protein in its capacity to induce DC activation and migration. 6xHistidin(His₆)-tagged recombinant full length mOPN was expressed by stable transfected human HEK-293-EBNA cells and mOPN was purified by its His₆-tag. Full-length mOPN was cleaved by thrombin and fragments were separated and purified by N-terminal His₆-tag. Sialic acid moieties were removed by neuraminidase. Analyzing the circular dichroism of each protein, we found the eucaryotic protein and its fragments to be unstructured in solution showing a spectrum characteristic for a random coil with a minimum at 200 nm, which is in accordance to findings for OPN expressed in E. coli. Investigating the function of the full length eucaryotic OPN, compared to E. coli derived GST-OPN, we found both forms to induce DC activation, as measured by their expression of MHC-II and CD86. Interestingly, the DC activating effect of eucaryotic OPN is more pronounced than the effect of bacteria derived GST-OPN. Chemotaxis assays with immature BM-DC were performed with full length OPN and the generated fragments demonstrated the eucaryotic OPN to be the more potent chemotaxin. The most important domain mediating DC-migration seems to be located within the C-terminal half of the molecule, which contains the CD44 binding domain, because the N-terminal fragment, with the RGD-sequence, was a less effective chemoattractant. Neuraminidase treatment did not alter OPN activity, indicating that the promigratory function of OPN on DC is not influenced by Sialic acid residues. In conclusion we found eucaryotic OPN to have a random coil structure in solution. Functionally, OPN cleavage, in vivo occurring at sites of inflammation may influence the outcome of an immune response by differentially modulating DC activation and migration.