The aim of this study was to assess structural and biochemical differences in the extracellular matrix of the fetal and adult porcine mitral heart valves in relation to their mechanical characteristics. Using tensile tests it was demonstrated that the material properties of porcine mitral heart valves progressively change with age. The collagen content of the adult heart valve, as estimated by hydroxyproline assay, increases three times as compared with fetal heart valves. Transmission electron microscopy demonstrated that the diameter of collagen fibrils increased in adult heart valves compared with fetal heart valves. The level of collagen cross-linking is lower in the fetal heart valve than the adult heart valve. The reported age differences in the material properties of fetal and adult porcine heart valves were associated with increases in collagen content, the diameter of collagen fibrils and the level of collagen cross-linking. These data lay a foundation for systematic elucidation of the structural determinants of material properties of heart valves during embryonic and postnatal valvulogenesis. They are also essential to define the desirable level of tissue maturation in heart valve tissue engineering.