Several families of synthetic polymers, including degradable poly(beta-amino ester)s, have been previously shown to effectively mediate gene transfer. However, the combined impact of potentially significant factors-such as polymer molecular weight, polymer chain end-group, and polymer/DNA ratio-on different gene transfer properties has yet to be systematically investigated. The elucidation of these relationships may aid in the design of nonviral vectors with greatly enhanced transfection properties. To examine these factors, two distinct poly(beta-amino ester) structures, Poly-1 and Poly-2, were generated by adding 1,4-butanediol diacrylate and 1,6-hexanediol diacrylate, respectively, to 1-aminobutanol. Twelve unique versions of each structure were synthesized by varying amine/diacrylate stoichiometric ratios, resulting in polymers with either amine or acrylate end-groups and with molecular weights ranging from 3350 to 18000. Using high throughput methods, all polymers were tested in quadruplicate at nine different polymer/DNA ratios ranging from 10:1 w/w to 150:1 w/w. Through the optimization of molecular weight, polymer chain end-group, and polymer/DNA ratio, these polymers successfully mediated gene transfer at levels that surpassed both PEI and Lipofectamine 2000 in vitro.