The purpose of this study was to analyze the potential of various types of biodegradable microspheres (MS) (i) to activate in vitro cell line-derived macrophages (RAW 264.7, Mphi), and primary peritoneal and bone marrow-derived mouse Mphi, to prolong the release and presentation of microencapsulated synthetic malaria antigens by Mphi after uptake of antigen-loaded MS, and (ii) to stimulate an immune response in mice against a microencapsulated synthetic malaria antigen. The MS were made of various types of poly(lactide-co-glycolide) (PLGA) or chitosan cross-linked with tripolyphosphate. PLGA, but not chitosan MS, were efficiently ingested by Mphi. Upon exposure to the various MS types, Mphi increased only the production of reactive oxygen intermediates (ROI), while the production of nitric oxide (NO), tumor necrosis factor alpha (TNF-alpha), and the expression of cyclooxigenase-2 (COX-2), inducible NO synthase (iNOS), the cell surface markers MHC class I and II, and CD 86 remained unaffected. In vitro release of the microencapsulated antigen from PLGA50:50 MS followed a pulsatile pattern and extended over 14 weeks. This prolonged antigen release was also mirrored in the significantly prolonged antigen presentation over more than 7 days by Mphi after uptake of antigen-loaded PLGA MS. Finally, antigen-loaded PLGA MS induced a solid immune response in mice after a single s.c.-injection, which was only slightly inferior to the antibody titers measured with the control formulation with Montanide ISA720. These results suggest that MS are well tolerated by Mphi. The prolonged antigen presentation by Mphi, as measured in vitro, along with the capacity to induce a strong immune response in animals emphasize that biodegradable MS are a very promising delivery system for both preventive and immunotherapeutic vaccines.