Gelatin, a natural macromolecule, is widely used in biomedical and biotechnological applications and is a good candidate for preparation of microspheres and microcapsules for the purpose of controlled release applications of drugs. In this study, gelatin microspheres crosslinked with glutaraldehyde with different crosslink densities were prepared by the phase separation technique induced by temperature change. The chemical structures of the microspheres were examined with FTIR and formation of crosslinks were observed. Topography, size, and shape of the microspheres were examined with scanning electron microscopy (SEM) and a narrow size distribution (approximately 1 mu m) was observed. Thermal properties of microspheres were analyzed by differential scanning calorimetry and an increase in glass transition temperature values with an increase in crosslinking was observed. For each sample, dry and swollen densities of microspheres were determined by pycnometric methods and the average molecular weights between the crosslinks, M(C), were calculated from the equilibrium swelling experiments using the modified Flory-Rehner thermodynamic theory. A decrease in percent solvent content values with decreasing average molecular weight between the crosslinks was observed.