Collagen type I fibrils, reconstituted in vitro in the presence of heparin, exhibit an unusually thick and straight shape. A detailed structural analysis by scanning force and scanning electron microscopy revealed a non-linear dependence in size distribution, width-to-length ratio, and morphology over a wide range of glycosaminoglycan (GAG) concentrations. By varying molecular weight, degree of sulphation, charge, and concentration of different GAGs we are able to correlate the morphological data with kinetic turbidimetric measurements, and quantitation of fibril-bound GAG. The experiments imply a pronounced impact of the prenucleation phase on the cofibril morphology as a result of the strong electrostatic interaction of heparin with tropocollagen. Heparin is assumed to stabilize the collagen microfibrils and to enhance their parallel accretion during cofibrillogenesis with preservation of the typical asymmetric collagen banding pattern. The heparin quantitation data show heparin to be intercalated as a linker molecule with one specific binding site inside the cofibrils. The reconstituted cofibrils with their unusual morphology and GAG intercalation-a phenomenon not reported in vivo-can be expected to exhibit interesting mechanical and biochemical behaviours as a biomaterial for extracellular matrix scaffolds.