Chitosan microspheres were prepared by an emulsion-phase separation technique without the use of chemical cross-linking agents; alternatively, ionotropic gelation was employed in a w/o emulsion. The possibility of three kinds of anions (tripolyphosphate, citrate and sulphate) to interact with chitosan was investigated by turbidimetric titration. The results indicate that there are electrostatic interactions between the above anions and chitosan in a certain region of solution pH (1.0-7.5 for sulphate/chitosan, 4.5-7.5 for citrate/chitosan and 1.9-7.5 for tripolyphosphate/chitosan), that is related to the natural characteristics of the anions. Out of the pH region where anions interact with chitosan, no microspheres were formed. However, even in the pH region where anions can interact with chitosan, only irregular microparticles were obtained in the case of the conventional emulsification and ionotropic gelation method, while spherical microspheres with diameters in the range of tens of microns were obtained when a modified process was employed. The key point of the modified process is the introduction of gelatin and allowing the ionic cross-linking process of chitosan/gelatin w/o emulsions to take place under coagulation conditions at a low temperature. The surface of sodium sulphate cross-linked chitosan/gelatin and sodium citrate cross-linked chitosan/gelatin microspheres was very smooth, but large gaps were observed on the surface of tripolyphosphate/chitosan microspheres. The increase of stirring speed led to a decrease in diameter and a narrowing in size distribution.