The stability of the complexes between DNA and nonviral vectors is a crucial parameter for efficient gene delivery into target cells. The stability must be high enough to prevent any dissociation during interaction with the plasma membrane but low enough to allow the dissociation that is required for efficient internalization into the nucleus. In this report, we investigated the stability of complexes of DNA with two cysteine surfactants (guanidinocysteine N-decylamide, C-10-CG+, and ornithinyl-cysteinyl-tetradecylamide, C-14-CO), able to convert themselves, via oxidative dimerization, into cationic cystine lipids. To this end, we determined the critical aggregation concentration (cac) and the binding constants of the surfactants for DNA by using the fluorescence quenching of the DNA his-intercalating agent, YOYO-1, that results from the dye clustering induced by the collapse of DNA. The cac's of C-10-CG+ and C-14-CO monomeric forms are 2.5 and 1 muM, respectively, and are slightly less than the 5 muM value for CTAB, taken as a model of nondimerizable surfactant. Dimerization of C-10-CG+ and C-14-CO reduces the cac to 400 and 1 nM, respectively. The strong stabilization induced by oxidation of C-14-CO is further confirmed by the increase in the rigidity of the micellelike domains in the complexes, as deduced from the rotational correlation time of the hydrophobic probe 1,6-diphenylhexatriene. In keeping with the stability data, no dissociation of the (C-14-CO)(2)/DNA complexes occurs in the presence of neutral vesicles (that mimic the external leaflet of the plasma membrane), while a significant dissociation was observed with (C-10-CG+)(2)/DNA complexes and an even larger one with CTAB/DNA complexes. Similarly, (C-14-CO)(2)/DNA complexes do not dissociate in the presence of anionic vesicles (that mimic the cytoplasmic leaflet of the plasma membrane), while a complete dissociation and DNA release occurs with both (C-10-CG+)(2)/DNA and CTAB/DNA complexes. Both the initial interaction with the plasma membrane and the release of DNA in the cytoplasm are strongly dependent on the stability of the complexes obtained with this new class of nonviral vectors.