Investigation was carried out to elucidate the influence of process parameters on the mean particle size of chitosan microspheres produced by water-in-oil (w/o) emulsion crosslinking method. The aqueous chitosan solution was dispersed in the oil phase composed of 40% (v/v) sunflower oil and 60% (v/v) n-hexadecane in the presence of Tween 80 surfactant. The droplets were solidified and hardened by glutaraldehyde crosslinking agent. The particle size of the obtained microspheres was influenced by four independent process variables, such as stirring rate, and the concentrations of chitosan, Tween 80 and glutaraldehyde. To study these effects, 4-factorial 3-level Box-Behnken type experimental design and statistical analysis of the experimental results were carried out in three consecutive steps. First, the 4 independent factors at 3 levels were taken into consideration without repetitions, resulting in 27 scheduled experiments. Second, the design was completed by further experiments to check the effects of some of the variables at five levels both inside and outside the original factor intervals used in the first step. In addition, five repeated experiments at the central points of the four factors were also involved in the second step to determine the pure error. In order to elucidate the effects of process variables on the mean particle size, non-linear multidimensional regression was determined. Third, the less significant effects were neglected resulting in a simplified model equation that proved to be suitable for the prediction of the mean particle size as a function of the studied process parameters. (c) 2008 Elsevier B.V. All rights reserved.