Oligonucleotides (ONs) such as antisense oligonucleotides (AS-ON) and siRNAs are used as experimental tools to study gene function and are currently being tested in clinical trials for use as therapeutic anticancer agents. However, their therapeutic use has been limited by their low physiological stability and their slow cellular uptake. The systemic delivery of sequence-specific AS-ON targeting the EWS/FLI1 gene product by a targeted, nonviral delivery system dramatically inhibits tumor growth in a murine model of Ewing's sarcoma. The nonviral delivery system uses a poly-iso-hexyl-cyanoacrylate (PIHCA)-containing polycation (chitosan) to bind and protect the AS-ON. No antitumor effect is observed using a control oligonucleotide sequence. We found here that injection of the free AS-ON stimulates tumor growth independently of its sequence and that this stimulation is abolished in the presence of nanosphere-chitosan, which exerts with the oligonucleotides a specific inhibitory effect on tumor growth. The stimulation of tumor growth is likely to be due to a polyanionic effect; indeed, a similar stimulatory response is observed upon treatment with dextran sulfate and heparin in vivo. These results suggest that ON loaded onto nanosphere-chitosan provides efficient and tumor-specific delivery, and provides protection against a polyanionic secondary effect.