Collagen, which is one of the most abundant proteins in mammalian proteomes, is of significant medical and also biotechnological importance. In tissue engineering, collagen is used as natural matrix to facilitate growth of mammalian cells in 3D-structures. Here, large amounts of highly purified Type I collagen have been obtained from rat tail tendon by a two-step purification, suitable for industrial up-scaling, involving extraction with 9 M urea followed by Superose 12 chromatography. Mass spectrometry identified only collagen Type I peptides indicating that the extracted collagen was homogeneous. Furthermore, a substantial amount of post-translational modifications were identified, including the presence of hydroxylated proline at the X-position in the G-X-Y repeats, which give new insight into collagen structure in vivo. The purified collagen was renatured quantitatively to form triple-helices by dialysis against water, as judged by UV-circular dichroism. This urea-extracted collagen (UC) was tested for its suitability as a matrix for growth of mammalian cells in tissue engineering. Cultures of a fibroblast cell line grown on urea-extracted collagen showed a higher motility and reduced stress levels than those grown on acetic acid-extracted collagen as determined by morphological studies and transcriptional analysis of selected marker genes using real time-PCR. These results indicate the suitability of the urea-extracted collagen obtained for biotechnological applications and tissue engineering. (C) 2009 Elsevier Ltd. All rights reserved.