This work describes the immobilization of beta-galactosidase onto polyelectrolyte multilayer assemblies of the polyanion poly[1-[4-(3-carboxy-4-hydroxyphenylazo)benzenesulfonamido]-1,2-ethanedi yl, sodium salt] (PAZO) and the polycation poly(ethylenimine) (PEI) constructed by electrostatic self-assembly (ESA). A single layer of beta-galactosidase was deposited over a precursor film comprising up to five bilayers of the PEI/PAZO polyelectrolyte pair. The enzyme was deposited on both the polycationic (PEI) and the polyanionic (PAZO) surfaces. Quartz crystal microbalance with dissipation monitoring (QCM-D), single-wavelength ellipsometry, and UV-visible absorption spectroscopy revealed differences in both the amount of beta-galactosidase incorporated in each of the multilayer assemblies and the resulting enzyme packing density in the films. The enzymatic films were immersed in a reaction solution containing o-nitrophenyl-beta-D-galactopyranoside (ONPG), and absorbance measurements were used to monitor the concentration of o-nitrophenyl (ONP), the product of the beta-galactosidase catalyzed by hydrolysis of ONPG. Although our data indicate that comparable amounts of beta-galactosidase are incorporated onto both surfaces, enzymatic activity is substantially inhibited when the beta-galactosidase is immobilized on the polyanionic surface compared to the enzyme on the polycationic surface. The difference in catalytic activities reflects the different abilities of the two polyelectrolytes to screen the protein's active site from the substrate environment. In both assemblies, the protein interpenetrated the PEI/PAZO multilayer, disrupting the J-aggregated state of the PAZO chromophores. This work demonstrates that the charge, conformation, and composition of underlying polyelectrolyte cushions have a significant effect on the structure and function of an immobilized protein within functional nanoassemblies.