A variety of molecular gradients of alkanethiols with the structure HS-(CH2)(m)-X (m = 15; X = COOH, CH2NH2, or CH3) and oligo(ethylene glycol)-terminated alkanethiols with the structures HS-(CH2)(15)-CO-NH-Eg(n) (n = 2, 4, or 6), HS-(CH2)(15)-CO-NH-Eg(2)-(CH2)(2)-NH-CO-(CH2)(4)-biotin, and HS-(CH2)(15)-CO-NH-Eg(6)-CH2-COOH were prepared on polycrystalline gold films. These gradients were designed to serve as model surfaces for fundamental studies of protein adsorption and immobilization phenomena. Ellipsometry, infrared spectroscopy, and X-ray photoelectron spectroscopy, operating in scanning mode, were used to monitor the layer composition, gradient profiles, tail group conformation, and overall structural quality of the gradient assemblies. The gradient profiles were found to be 4-10 mm wide, and they increased in width with increasing difference in molecular complexity between the thiols used to form the gradient. The oligo(ethylene glycol) thiols are particularly interesting because they can be used to prepare so-called conformational gradients, that is, gradients that display a variation in oligo(ethylene glycol) chain conformation from all trans on the extreme Eg(2,4) sides, via an amorphous-like phase in the mixing regimes, to helical at the extreme Eg(6) sides. We demonstrate herein a series of experiments where the above gradients are used to evaluate nonspecific binding of the plasma protein fibrinogen, and in agreement with previous studies, the highest amounts of nonspecifically bound fibrinogen were observed on all-trans monolayers, that is, on the extreme Eg(2,4) sides. Moreover, gradients between Eg(2) and a biotinylated analogue have been prepared to optimize the conditions for the immobilization of streptavidin. Ellipsometry and infrared spectroscopy reveal high levels of immobilization over a fairly broad range of compositions in the gradient regime, with a maximum between 50 and 60% of the biotinylated analogue in the monolayer. A pI gradient composed of (NH3+/COO-)-terminated thiols was also prepared and evaluated with respect to its ability to separate differently charged proteins, pepsin, and lysozyme, on a solid surface.