To improve gene transfer activity of a new nonviral vector, a polyamidoamine dendrimer (G2) conjugate with alpha-cyclodextrin (alpha-CDE conjugate (G2)), we prepared alpha-CDE conjugates with dendrimer having different generations (G3 and G4), and their gene transfer activities were compared with those of alpha-CDE conjugate (G2) and TransFast, a novel transfection reagent. alpha-CDE conjugates (G2, G3, and G4) formed the complexes with pDNA, changing the zeta-potential and particle size of pDNA complexes and the protection of pDNA from DNase I in a charge ratio-dependent manner, although their differences at higher charge ratios (vector/pDNA) were small. The gene transfer activity of alpha-CDE conjugates (G2, G3, and G4) was higher than that of the corresponding dendrimer alone in NIH3T3 and RAW264.7 cells. Of these CDE conjugates, alpha-CDE conjugate (G3) had a superior gene transfer activity which was comparable to that of TransFast in NIH3T3 cells. The intracellular distribution of pDNA after application of the pDNA complex with alpha-CDE conjugate (G3) to NIH3T3 cells was different from that with dendrimer alone (G3), although the cellular association of pDNA was almost comparable among all vectors. alpha-CDE conjugate (G3) strongly interacted with a fluorescence probe, 2-(p-toluidinyl)-naphthalene-6-sulfonate (TNS), suggesting that the conjugate possesses the inclusion ability with biomembrane constituents such as phospholipids after transfection. These results suggest that alpha-CDE conjugates, particularly the G3 conjugate, could be novel nonviral gene transfer agents.