The aim of this study was to compare two methods to encapsulate a 25-mer-phosphorothioate oligonucleotide (ODN) into poly(D,L-lactic acid) (PLA) particles. Antisense oligonucleotides belong to a new therapeutic class especially attractive for the treatment of cancers and viral diseases. The development of these new drugs suffers, however, from poor stability in biological media and very low cellular uptake. Polymeric particulate systems display interesting features for ODN delivery. ODN are highly hydrophilic and most encapsulation methods are inappropriate for such molecules. Using poly(D,L-lactide) polymer, two methods of encapsulation were compared. First, a double emulsion technique was used to prepare nano- and microparticles. Secondly, the ODN was combined with a quaternary ammonium, the cethyltrimethyl-ammonium bromide (CTAB), to enhance the hydrophobicity of the molecule before entrapment by the emulsification-diffusion method. Both methods led to the formation of individualized and spherical particles loaded with a significant amount of ODN. Similar entrapment efficiencies were obtained for the nanoparticles prepared by both methods (approx. 27% of the theoretical loading) whereas 45% of entrapment efficiency was observed for the microparticles. Seventy five percent of the ODN were released in 60 min with the particles prepared by the emulsification-diffusion method, whereas only 7% were released in 60 h when using the double emulsion method. A viability test on U-937 cells showed better survival rates with the particles prepared by the double emulsion technique. The results suggest that the location of the ODN in the polymeric matrix is affected by the encapsulation method. Particles containing CTAB appeared more toxic than the ones obtained by the double emulsion technique, however, these particles can still be used for antisense activity since high oligonucleotide loading can be achieved.