This project was aimed at illustrating the potential Use of poly-DL-lactide-poly(ethylene glycol) (PELA) microspheres as a hepatitis B surface antigen (HBsAg) delivery system following subcutaneous (s.c.) or oral immunization over the current injection of an alum-absorbed antigen. The antigen-loading microspheres were elaborated by the solvent-extraction method based on the formation of modified multiple w/o/w, emulsion. The microspheres were characterized by their particle size, HBsAg entrapment, and in vitro HBsAg release behavior. Balb/c mice were immunized with an s.c. injection and oral administration of a single dose and two doses of a microsphere formulation. For comparison, the alum-absorbed HBsAg-immunized mice had a following intramuscular (i.m.) injection at weeks 0 and 4. At weeks 8, 10, 14, and 24 postadministration, the blood and saliva samples were collected and detected by the enzyme-linked immunosorbed assay (ELISA) method. A single injection of HBsAg/PELA microspheres could induce a serum IgG antibody level comparable to the two-injection alum-absorbed HBsAg at the 14th week and higher than that at the 24th week. The saliva IgA of peroral groups was significantly higher than that of the s.c. injection of a microsphere formulation and i.m. injection of soluble antigen. These preliminary results demonstrated the potential of oral administration of antigen-loading microspheres in the induction of a secretary immune response, and it is suggested that a single-dose s.c. injection of antigen-loading microspheres would be an efficient immunization schedule to elicit a protective response.