γ-Irradiation is currently the method of choice for terminal sterilization of drug delivery systems made from biodegradable polymers. However, the consequences of γ-sterilization on the immune response induced by microencapsulated antigens have not yet been reported in the literature. The aim of the present work was to evaluate the effect of γ-irradiation on the biopharmaceutical properties of PLGA microspheres containing SPf66 malarial antigen. Microspheres were prepared by a (w/o/w) double emulsion/solvent extraction method. Once prepared, part of the formulation was irradiated at a dose of 25 kGy using 60Co γ as radiation source. The in vitro results obtained showed that the γ-irradiation exposure had no apparent effect on SPf66 integrity and formulation properties such us morphology, size and peptide loading. Only the release rate of SPf66 was slightly faster after γ-irradiation. Subcutaneous administration of irradiated and non-irradiated microspheres into mice induced a similar immune response (IgG, IgG1, IgG2a levels) and was comparable to that obtained with SPf66 emulsified with Freund's complete adjuvant. These observations illustrate the applicability of γ-irradiation as a method of terminal sterilization of microparticulate delivery systems based on chemically synthesized antigens encapsulated into biodegradable PLGA microspheres.