Background: Nonviral polymeric delivery systems are explored to enhance clinical development of nucleic acids as therapeutic entities for effective management of debilitating conditions such as cancer. This study was to compare safety and efficacy of quaternary amine-containing methacrylate polymer Eudragit® RL PO (ERL) and poly[N-(2-hydroxypropyl)methacrylamide]-poly(N,N-dimethylaminoethyl methacrylate) copolymer (pHPMA-b-pDMAEMA), which contains secondary and tertiary amines, as effective gene carriers. Methods: Polyplexes of pAcGFP1-C1 with ERL or pHPMA-b-pDMAEMA were fabricated at different N/P ratios. Formation of DNA/catiomer nanostructures was monitored by ethidium bromide intercalation and agarose gel retardation. Particle size, zeta potential and cytotoxicity of different polyplexes were characterized. Transfection efficiency in presence and absence of serum was assessed using confocal microscopy. Results: pHPMA-b-pDMAEMA demonstrated at least a 10-fold greater DNA condensation capacity per weight unit than ERL. However, DNA intercalation with pHPMA-b-pDMAEMA was reduced in presence of serum-free cell culture media, whereas polyplex formation with ERL was equivalent in phosphate-buffered saline, pH 7.4 and serum-free cell culture media. Cellular safety of HeLa cells was not compromised by polyplexes fabricated with either polymer up to N/P=4. However, ERL alone was more toxic. In absence of serum, pHPMA-b-pDMAEMA polyplexes at N/P=4 induced equivalent transgene expression as control TurboFect™ polyplexes. In contrast, ERL-containing nanoassemblies failed to produce measurable transgene expression. Inclusion of serum significantly decreased transfection efficiency of pHPMA-b-pDMAEMA-containing polyplexes by ~30% at N/P=4 and ~50% at N/P=2. Conclusion: Polyplexes fabricated with secondary and tertiary amine-containing pHPMA-b-pDMAEMA copolymer represent more effective gene delivery systems than nanoassemblies composed of quaternary amine-containing ERL and should be further explored for clinical applications.