The adsorption of avidin, neutravidin, and biotin-labeled dextran 10,000, dextran 70,000, bovine albumin, and insulin to the gold electrodes of thickness-shear-mode acoustic wave devices was studied in a flow injection analysis configuration. Electrodes with different surface rugosity and free energy were also examined. Changes in series resonance frequency for the various molecules as a result of electrode adsorption cannot be explained in terms of the conventional mass-response effect. Perturbation of the properties of the protein and polysaccharide layers, such as their viscosity, and acoustic coupling phenomena offer a more reasonable basis to evaluate the results. In an analogous fashion, the acoustic wave sensor signals obtained for the formation of the various avidin- and neutravidin-biotin conjugate samples do not correspond to the additional mass deposited on the device surface. The responses for the binding of biotinylated insulin to avidin and to the nonglycosylated form of the parent molecule were completely different. This result is ascribed to a difference of surface free energy associated with the two protein-based interfaces. The acoustic wave sensor was also employed to investigate the formation of avidin-biotin conjugate multilayer structures on the surface of the device. Multilayer buildup is detectable for several pairs of avidin-biotin interactions, but the corresponding shifts in series resonance frequency gradually diminish in magnitude. Finally, the introduction of biotin itself appears to result in structural changes in the multilayer deposit, causing alterations in viscoelastic properties.