The sol-gel encapsulation process has been exploited in recent years for the immobilization of proteins to be used as biosensors. Sol-gels derived from tetramethyl orthosilicate provide a stable environment for the macromolecule combined with the free flow of small substrates to a protein＇s binding site. The functionality of a number of enzymes within the solid matrix has been demonstrated. However, very little biophysical characterization of the encapsulated proteins has been done. in this study, time-resolved fluorescence anisotropy was used to compare the rotational mobility of two probes in sol-gel matrices derived from three different preparative methods. A small fluorescent probe, sulforhodamine 101 (SR101), was used to gauge the relative solvent viscosity within the sol-gels. Magnesium protoporphyrin IX substituted myoglobin (MgMb) provides a convenient fluorescent probe for measuring rotational dynamics of a typical globular protein. The anisotropy decay of the Mg-heme is sensitive only to the global protein motion. The SR101 reveals both low (phi < 1 ns) and high (phi = 6-500 ns) viscosity encapsulation sites within the matrix, and the populations of these sites are dependent on gel preparation and age. The protein, however, shows greatly diminished decay of the fluorescence anisotropy (phi similar to 1 mu s) in two of the three gels (but was denatured in the third). This is consistent with restrictive encapsulation sites where size and/or environment substantially impedes rotational diffusion.