Mechanical properties of polyelectrolyte multilayer films were studied by nanoindentation using the atomic force microscope (AFM). Force-distance measurements using colloidal probe tips were systematically obtained for supported films of poly(L-lysine) and hyaluronan that are suited to bio-application. Both native and covalently cross-linked films were studied as a function of increasing layer number, which increases film thickness. The effective Young's modulus perpendicular to the film, E-perpendicular to, was determined to be a function of film thickness, cross-linking, and sample age. Thick PEM films exhibited a lower E-perpendicular to than thinner PEM, whereas the Young's modulus of cross-linked films was more than 10-fold larger than native films. Moduli range from approximate to20 kPa for native films up to approximate to800 kPa for cross-linked ones. Young's moduli increased slightly with sample age, plateauing after similar to4 weeks. Spreading of smooth muscle cells on these substrates with pre-attached collagen proved to be highly dependent on film rigidity with stiffer films giving greater cell spreading.