Using the Langmuir-Blodgett (LB) technology we have pioneered a straightforward and low-cost approach to fabricate highly oriented collagen in thin film format (thickness: similar to 20nm, surface areas: 2.5 x similar to 6.0 cm). An important factor for the use of these films is their cohesion under various conditions. Film formation was studied by coating hydrophilic or hydrophobic glass substrates. The fresh or aged (2 1/2 years, 18 degrees C.) collagen solutions used for this purpose either contained collagen network-stabilizing agents (n-propanol or phosphate ions) or were prepared without these stabilizers. Film formation on the air/water interface was analyzed by pressure-area isotherms. Maximum surface pressures were similar to 4-7mN/m and similar to 10-18 mN/m for isotherms using n-propanol or phosphate buffer saline (PBS), respectively, versus similar to 0.4-0.8 mN/m without using a stabilizer; with higher surface pressures for the combination fresh solution/n-propanol or aged solution/PBS. Deposited films were studied by optical and electron microscopy and fast Fourier transform analysis. Coatings (to both substrate types) exhibit a defined orientation of collagen aggregates within a matrix of oriented collagen when freshly made or aged collagen solutions were used and n-propanol was present during film formation. The higher degree of hydrophilicity of the aged solution does not adversely affect the cohesion and collagen orientation during film formation. Using physiological phosphate ions shows that deposition of defect-free and oriented collagen (on both substrate types) is only possible using fresh collagen solutions. Unlike n-propanol-containing solutions, films were most stable using hydrophilic glass substrates. Film formation failed in the absence of network stabilizers. Controlling the cohesion via (a) the water accessibility of collagen structures, (b) specific network stabilizers and (c) substrate properties enables tunable film characteristics for future biomedical approaches.