Polyamidoamine (PAMAM) dendrimer represents one of the most efficient polymeric gene carriers. Toinvestigate the effect of the core structure and generation of dendrimers on the complex formation andtransfection efficiency, a series of PAMAM dendrimers with a trimesyl core (DT) at different generations(DT4 to DT8) were developed as gene carriers and compared with the PAMAM dendrimers derived frompentaerythritol (DP) and inositol (DI). The minimal generation number of DTs at which the dendrimer hasenough amino group density to effectively condense DNA was higher (generation 6) than those of DPs andDIs (generation 5). DTs of generation 6 or higher condensed DNA into complexes with an average diameterranging from 100 to 300nm, but the 4th and 5th generations of DT (DT4 and DT5) formed only a severeaggregate with DNA. Interestingly, the DT6/pDNA complex was determined to be much smaller (100-300nm) than those prepared with DP5 or DI5 (>600 nm) at N/P ratios higher than 15. The optimal generationnumbers at which the dendrimers showed the highest transgene expression in COS-7 cells were 5 for DPsand DIs but 6 for DTs. The DT6/pDNAcomplex with smaller size mediated higher transgene expression inCOS-7 cells than those prepared with DP5 or DI5. The in vitro transfection efficiency of the DT dendrimersas evaluated in HeLa cells, COS-7 cells, and primary hepatocytes decreased in the order of DT6 > DT7 >DT8 > DT5 > DT4. The transfection mediated by DT6 was significantly inhibited by bafilomycin A1. Theacid-base titration curve for DT6 showed high buffer capacity in the pH range from 5.5 to 6.4 (pKa-6).This permits dendrimers to buffer the pH change in the endosomal compartment. However, the transfectionefficiency mediated by DT6 decreased significantly in the presence of serum in both HeLa cells and COS-7cells. The cytotoxicity of DTs evaluated in HeLa cells using the 3-4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide assay showed a trend of increasing toxicity with the polymer generations. TheLD50 values of DT4 through DT8 were 628, 236, 79, 82, and 77 íg/mL, respectively, which were higherthan that of poly(ethyleneimide) (18 íg/mL) and poly(L-lysine) (28 íg/mL) in the same assay. With a lowercytotoxicity and versatility for chemical conjugation, these PAMAM dendrimers with a DT core warrantfurther investigation for nonviral gene delivery.