The objective of this study was to produce enteric microspheres containing bovine insulin as a model drug using a water-in-oil-in-water (w/o/w) emulsion solvent evaporation method, and the preparative conditions were optimized. When hydroxypropylmethylcellulose acetate succinate (AS-HG type; high content of succinyl group) was employed as an enteric wall material, optimized microspheres showed almost 90% of the loading efficiency of insulin and 30.8 microns of mean volume diameter. The mixture of methylene chloride and acetone (4:1) as an oleaginous phase, 400 microliters of bovine insulin solution (dissolved in 30% of acetic acid) as an internal aqueous phase, and 1.0% of polyvinylalcohol dissolved in pH 3.0 citrate buffer as an external aqueous phase, were employed in the experiment. In relation to other enteric cellulose derivatives (AS-MG type, AS-LG type; medium and low content of succinyl group, respectively), the microencapsulation using a simultaneous preparation method also resulted in quite high loading efficiencies, whereas the choice of poly(methyl methacrylate) as a wall material caused aggregation or flocculation in the preparative process of every batch. The AS-HG microspheres showed very fast release profile in pH 6.8 buffer, but no released fraction was observed in pH 1.2 buffer. This phenomenon suggested enteric characteristics of prepared microspheres. Finally AS-HG microspheres containing 4% lauric acid and 9% insulin were prepared, suspended in 0.1% of carboxymethyl cellulose solution, and administered to the rat rectum (corresponding to 50 I.U./kg insulin). The plasma glucose level reached minimum level at 0.5 h after administration then gradually rose to normal.