Natural membrane vesicles (n-MVs) produced by 'Pseudomonas aeruginosa ' PAO1 and PAO1 carrying plasmid pAK1900 (p-MVs) were analyzed for DNA content. PCR analysis using the purified MVs as template indicated that only plasmid DNA is contained within p-MVs. p-MVs could fuse into the outer membrane (OM) of PAO1 and 'Escherichia coli' DH5[alpha], delivering the plasmid into the periplasm where it would only have to by-pass the plasma membrane (PM) for effective transformation. However, p-MVs did not transform PAO1 nor DH5[alpha] under a variety of transforming conditions. To ensure that MV-encapsulated pAK1900 was not derived from lysed cells, the physical and ultrastructural differences between both n- and p-MVs were determined. By isopycnic sucrose density gradients, cryo-TEM, characterization of the OM protein and LPS profiles, and enzyme analysis, p-MVs were found to be OM-based. Studies examining the internalization of DNA within MVs suggested that this incorporation could occur by two routes; (1) the MV encapsulates periplasmic DNA and (2) exogenous DNA can be internalized within the MV.