A simple and in situ method, by using a high-voltage electrostatic system, for the fabrication of chitosan microspheres (in a form of isolatable microgels) by an extrusion process, exhibiting variable sizes and different membrane structures, was presented. The chitosan microspheres exhibited good sphericity and were in the range of 185.8 +/- 13.8 to 380.9 +/- 11.5 mum in diameter. There were two significant factors, the pump flow rate and electrostatic field strength, that affected the chitosan microsphere size. The microsphere size decreased when the flow rate was increased from 0.1 to 0.4 mL/h. Also, the microsphere size decreased when the electrostatic field strength was increased from 5.5 to 6.5 kV/cm. However, when the electrostatic field strength was raised to 7 kV/cm and higher, the microsphere size increased. For the latter case, with other parameters fixed, chitosan microsphere size can be controlled by adjusting the electrostatic field strength and predetermined by a simple linear regression equation: Microsphere Diameter (D, in mum) = -(75.48) + 45.67 x (Electrostatic Field Strength, E, in kV/cm), at [7 less than or equal to (Electrostatic Field Strength) less than or equal to 10] (R-2 = 0.956, P < 0.001). Following treatment with various ratios of crosslinking/gelating (Na5P3O10/NaOH) agents, the prepared chitosan microspheres exhibited distinct membrane structures that yielded various mechanical strengths. In the Na5P3O10/NaOH ratio of 19, the chitosan microspheres had a distinct two-layer structure. The selection of crosslinking/gelating ratio provided an additional degree of freedom, permitting the simultaneous regulation of mechanical properties and permeability of the microspheres, without extra manipulation, and thus, improved applicability in the biomedical field. When the chitosan microsphere extrusion process was used to encapsulate (beta-tricalcium phosphate powder for application as bony material, we found that the ultra fine (beta-tricalcium phosphate powder was trapped inside of the membrane very well. After appropriate collecting procedures, stored microspheres also retained good spherical shape. (C) 2004 Wiley Periodicals.