Polyphosphazenes bearing cationic moieties were synthesized from poly(dichloro)phosphazene, which in turn was obtained by thermal polymerization of hexachlorocyclotriphosphazene in 1,2,4-trichlorobenzene. Next, either 2-dimethylaminoethanol (DMAE) or 2-dimethylaminoethylamine (DMAEA) side groups were introduced by a substitution reaction. The polymers were purified by dialysis against water and tetrahydrofuran, lyophilized and evaluated as polymeric transfectants. The polyphosphazenes were able to bind plasmid DNA yielding positively charged particles (polyplexes) with a size around 80 nm at a polymer/DNA ratio of 3:1 (w/w). The polyphosphazene-based polyplexes were able to transfect COS-7 cells in vitro with an efficiency comparable to a well-known polymeric transfectant [poly(2-dimethylaminoethyl methacrylate), pDMAEMA]. The toxicity of both polyphosphazenes was lower than pDMAEMA. The transfection efficiency for the poly(DMAE)phosphazene-based polyplexes was about threefold higher in the absence of serum than in the presence of 5.0% fetal bovine serum. This is probably caused by unfavorable interactions of the polyplexes with serum proteins. In contrast, the poly(DMAEA)phosphazene-based polyplexes showed a threefold lower transfection activity in the absence of serum. For this system, serum proteins likely masked the toxicity of the polyplexes, as shown by the XTT cell viability assay and confocal laser scanning microscopy studies. Preliminary degradation studies indicate that the polymers were indeed degradable. The half-life at pH 7.5 and 37 degrees C was around 7 days for poly(DMAE)phosphazenes and 24 days for poly(DMAEA)phosphazenes. This study shows that polyphosphazenes are a suitable and promising new class of biodegradable polymeric carriers for gene delivery.