The objective of this study was to establish the optimum conditions for preparing stable oil-in-water emulsions containing droplets surrounded by surfactant-chitosan layers. A primary emulsion containing small droplets (d(32) approximate to 0.3 mu m) was prepared by homogenizing 20 wt% corn oil with 80 wt% emulsifier solution (20 mM SDS, 100 mM acetate buffer, pH 3) using a high-pressure valve homogenizer. The primary emulsion was diluted with chitosan solutions to produce secondary emulsions with a range of oil and chitosan concentrations (0.5-10 wt% corn oil, 0-1 wt% chitosan, pH 3). The secondary emulsions were sonicated to help disrupt any droplet aggregates formed during the mixing process. The electrical charge, particle size, and amount of free chitosan in the emulsions were then measured. The droplet charge changed from negative to positive as the amount of chitosan in the emulsions was increased, reaching a relatively constant value (approximate to+50 mV) above a critical chitosan concentration (C-Sat), which indicated that saturation of the droplet surfaces with chitosan occurred. Extremely large droplet aggregates were formed at chitosan concentrations below C-Sat, but stable emulsions could be formed above C-Sat provided the droplet concentration was not high enough for depletion flocculation to occur. Interestingly, we found that stable multilayer emulsions could also be formed by mixing chitosan with an emulsion stabilized by a nonionic surfactant (Tween 20) due to the fact the initial droplets had some negative charge. The information obtained from this study is useful for preparing emulsions stabilized by multilayer interfacial layers.