Chitosan microspheres containing phenobarbitone were successfully prepared by glutaraldehyde cross-linking of an aqueous acetic acid dispersion of chitosan in light liquid paraffin containing sorbitan mono-oleate as a stabilizing agent. Uniform and spherical microspheres, with a loading efficiency up to 57.2%, could be prepared depending on the preparation conditions. The main parameters affecting the preparation and the performance of the prepared microspheres were the molecular weight and concentration of chitosan as well as the concentration of the used stabilizing agent. The incorporation of citric acid into the microspheres was found to increase the formation of a water-soluble gel when the microspheres come in contact with the dissolution medium increasing the rate of drug release. The particle size was shifted towards smaller diameters with increased concentration of sorbitan mono-oleate, up to 4.0% v/v, by use of a lower concentration of chitosan (1.0% w/v) and chitosan with low molecular weight. Rapid initial drug release (20-30% of the incorporated drug) was exhibited in all the prepared microspheres followed by slow release of the remaining amount of the drug. The release rate of the drug from the microspheres prepared from high molecular weight chitosan was slow in comparison with that prepared from medium and low molecular weight chitosan. High concentrations of sorbitan mono-oleate increased the rate of drug release.