Here we describe three novel collagen VI chains, ␣4, ␣5, and ␣6. The corresponding genes are arranged in tandem on mouse chromosome 9. The new chains structurally resemble the collagen VI ␣3 chain. Each chain consists of seven von Willebrand factor A domains followed by a collagenous domain, two C-terminal von Willebrand factor A domains, and a unique domain. In addition, the collagen VI ␣4 chain carries a Kunitz domain at the C terminus, whereas the collagen VI ␣5 chain contains an additional von Willebrand factor A domain and a unique domain. The size of the collagenous domains and the position of the structurally important cysteine residues within these domains are identical between the collagen VI ␣3, ␣4, ␣5, and ␣6 chains. In mouse, the new chains are found in or close to basement membranes. Collagen VI ␣1 chain-deficient mice lack expression of the new collagen VI chains implicating that the new chains may substitute for the ␣3 chain, probably forming ␣1␣2␣4, ␣1␣2␣5, or ␣1␣2␣6 heterotrimers. Due to a large scale pericentric inversion, the human COL6A4 gene on chromosome 3 was broken into two pieces and became a non-processed pseudogene. Recently COL6A5 was linked to atopic dermatitis and designated COL29A1. The identification of novel collagen VI chains carries implications for the etiology of atopic dermatitis as well as Bethlem myopathy and Ullrich congenital muscular dystrophy.Members of the collagen protein superfamily play important roles in maintaining extracellular matrix structure and function. To date 28 family members are known (1, 2), among which the fibril-forming collagens and the FACIT collagens form large subgroups. In addition, several collagens exist that have highly specific functions. Among these, collagen VI forms a distinct network of microfibrils in most connective tissues. Electron microscopy revealed a beaded filament structure of the microfibrils (3). The ␣1, ␣2, and ␣3 chains of collagen VI form heterotrimeric monomers that already intracellularly assemble to dimers and tetramers (4, 5). After secretion, filaments are formed by end to end interactions of the preassembled tetramers.The three previously known collagen VI chains contain a relatively short collagenous domain of about 335 residues together with VWA 3 domains, which are the characteristic non-collagenous domains of collagen VI. A common feature of VWA domains is their involvement in the formation of multiprotein complexes (6). Whereas all three collagen VI chains contain two C-terminal VWA domains, the ␣1 and ␣2 chains carry only one and the ␣3 chain ten VWA domains at the N terminus (7,8). In addition, the ␣3 chain contains a unique domain with similarities to salivary gland proteins, a fibronectin type III repeat, and a bovine pancreatic trypsin inhibitor/ Kunitz family of serine protease inhibitor domain (Kunitz domain) at the C terminus (8). It was suggested that the VWA domains play a role in the assembly of collagen VI (9 -11). However, recently the analysis of lysyl hydroxylase 3-deficient mouse embryos indicated...