Recent studies have indicated that there are many barriers to successful systemic gene delivery via cationic lipid vectors using the intravenous route. The purpose of this study was to investigate the effect of binding and interaction between erythrocytes, a major constituent of blood cells, and the complexes, in relation to the role of the helper lipid, on the in vivo gene delivery to the lung following intravenous injection. We used three types of cationic lipid vectors, DNA-DOTMA/Chol liposome complexes, DNA-DOTMA liposome complexes, and DNA-DOTMA/DOPE liposome complexes. Although the three types of vectors bind to murine blood cells in vivo and in vitro, DOTMA/Chol and DOTMA complexes with a higher in vivo transfection activity do not induce fusion between erythrocytes, whereas DOTMA/DOPE complexes, a less efficient vector in vivo, induce fusion between the erythrocytes after a short incubation period. Pre-incubation of DOTMA/DOPE complexes with erythrocytes significantly reduced the transfection efficiency while DOTMA/Chol- and DOTMA complexes were more resistant to such treatment. The differences in the physicochemical and structural properties of these complexes could explain the differences in interaction with erythrocytes and subsequent gene expression. Lipids in DOTMA/Chol and DOTMA complexes have a stable lamellar structure. However, lipids in DOTMA/DOPE complexes have a highly curved structure with high fluidity. These results indicate that the interaction with erythrocytes depends on the properties of the cationic lipid vectors and this is an important factor for intravenous gene delivery using cationic lipid vectors.