Static time-of-flight secondary ion mass spectrometry (ToF-SIMS), monochromatized x-ray photoelectron spectroscopy (XPS) and 125 I radiolabeling have been used to characterize albumin films adsorbed onto titanium, gold, polytetrafluoroethylene and r.f. glow discharge-deposited tetrafluoroethylene (TFE) substrates. A comparison between albumin and fibronectin films also was made. The intensities of characteristic amino acid mass fragments (immonium ions) detected in the static ToF-SIMS experiments depended on the protein type, the substrate type and the adsorption conditions, demonstrating the sensitivity of static ToF-SIMS for probing the structure of adsorbed protein films. Based on the results from albumin and fibronectin, static ToF-SIMS can provide information about the identity of adsorbed proteins and their conformation, orientation, denaturation, etc. X-ray photoelectron spectroscopy can distinguish pure protein films, but the higher molecular specificity of static ToF-SIMS is more useful than XPS for examining complex protein films. The 125 I radiolabeling experiments and the XPS atomic percentage of nitrogen were used to quantify the amount of adsorbed protein.