AbstractMembers of the Wnt protein family play roles in many aspects of embryogenesis and homeostasis. Despite their biological significance, characteristics of Wnt proteins still remain unclear, mainly due to their insolubility after the removal of serum. Here we examine Wnt proteins in serum-containing media by using analytical ultracentrifugation with a fluorescence detection system. This analysis reveals that Wnt3a assembles into high-molecular-weight complexes that become dissociable by interaction with the extracellular domain of the Frizzled8 receptor or secreted Wnt-binding protein sFRP2. Cross-linking and single-particle analyses of Wnt3a fractionated by gel filtration chromatography show the homo-trimer to be the smallest form of the assembled Wnt3a complexes. Fluorescence correlation spectroscopy and immunohistochemistry reveal that the assembly of Wnt3a complexes restricted their diffusion and signaling range inXenopus laevisembryos. Thus, we propose that the Wnt diffusion range can be controlled by a balance between the assembly of Wnt complexes and their dissociation.