The biophysical characteristics of vascular tissues are dependent to a large extent on the properties of their main structural components, fibrillar collagens. Stabilization of fibrillar collagens occurs initially through formation of difunctional, lysyl oxidase mediated cross-links whereby lysine or hydroxylysine residues in the non-helical portions of the molecule (telopeptides) are converted to aldehydes which then condense with hydroxylysine and other residues in neighbouring molecules to form intermolecular bonds. These borohydride reducible bonds are intermediates that are converted during maturation to trifunctional cross-links which include pyridinium, pyrrole and histidine-containing compounds [1]. Whether lysine or hydroxylysine residues are present in the telopeptides is crucial in directing the pathway of cross-link formation in a tissue-specific manner, so that complete hydroxylation of telopeptide lysine leads to pyridinium cross-link formation whereas collagen lacking telopeptide hydroxylysine forms histidine adducts of the difunctional bonds. The hydroxylation of telopeptide lysine is accomplished intracellularly by a separate lysyl hydroxylase enzyme to that which hydroxylates lysine residues destined to be in the helix. This enzyme, the long form of lysyl hydroxylase-2, is, therefore, the primary control for the pattern of lysyl oxidase-mediated cross-linking in collagen [2]. Functionally, the importance of these lysine-derived cross-links is well demonstrated by the effects of the lathyrogen, β-aminoproprionitrile, which, as an irreversible inhibitor of lysyl oxidase, results in the assembly of collagen fibrils lacking tensile strength because the pathways to cross-link formation are blocked. The differential effects of the various lysine-derived cross-links are not well established, although it is clear that the transformation from intermediate to mature forms is accompanied by increased inter-microfibrillar bonding, a feature that results in transverse arrays of cross-links across the fibril giving resistance to shear stresses. A second type of cross-linking mechanism that may also involve collagen lysine residues, but which should not be confused with lysyl oxidase-mediated processes, is that involving glycation or lipid oxidation products. Glycation has been the focus of much research and the importance in vascular biology of Advanced Glycation Endproducts (AGEs) as well as their receptor is increasingly recognised. In contrast to the maturation of lysyl oxidase-mediated cross-links, formation of AGEs in collagen is a true ageing process that is generally deleterious to the function of the tissue. Another important difference for age-related cross-linking is that these bonds are formed between collagen helices and do not involve the telopeptides. This leads to major functional consequences in terms of tissue stiffness and increased resistance to enzymatic degradation. The major effect of inter-helical cross-links on enzyme degradation of collagen is exemplified in skin collagen where increasing concentrations of an inter-helical, histidine adduct cross-link is matched by increased resistance to solubilisation by pepsin [3]. The structure and biochemistry of the many AGE compounds described to date will not be discussed in detail as this is the topic of another presentation in the present symposium. In many cases, however, the amounts of specific forms of these compounds in collagen are too low to have any functional significance, and compounds such as pentosidine should be viewed as markers of an ageing process rather than themselves being significant cross-links. Concentrations of the lysine-arginine cross-link, glucosepane, do, however, approach those of the lysyl oxidase-mediated cross-links in skin collagen of the elderly [4]. The properties of cardiovascular tissue are dependent on many factors and, in addition to collagen cross-linking, it is important to be able to assess the contribution of different collagen types as well as measure the other principal constituents of the extracellular matrix, elastic tissue and proteoglycans. New techniques being developed to accomplish these aims will be outlined, including cross-link analyses by mass spectrometry using multiple reaction monitoring, a procedure that overcomes problems in measuring collagen cross-links using conventional methods caused by the presence of reduced elastin-derived components. The overall aim of these studies is to provide information on how changes in the extracellular matrix affect the properties of both normal and diseased cardiovascular tissues.