A novel synthetic gene transfer vector was evaluated for tumor cell-specific targeted gene delivery. The folate receptor is a tumor marker overexpressed in more than 90% of ovarian carcinomas and large percentages of other human tumors. Folic acid is a high affinity ligand for the folate receptor that retains its binding affinity upon derivatization via its gamma carboxyl. Folate conjugation, therefore, presents a potential strategy for tumor-selective targeted gene delivery. In the current study, we investigated a series of folate conjugates of the cationic polymer polyethylenimine (PEI) for potential use in gene delivery. A plasmid containing a luciferase reporter gene (pCMV-Luc) and the folate receptor expressing human oral cancer KB cells were used to monitor gene transfer efficiency in vitro. Transfection activity of polyplexes containing unmodified polyethylenimine was highly dependent on the positive to negative charge (or the N/P) ratio. Folate directly attached to PEI did not significantly alter the transfection activity of its DNA complexes compared to unmodified PEI. Modification of PEI by polyethyleneglycol (PEG) led to a partial inhibition of gene delivery compared to unmodified PEI. Attaching folates to the distal termini of PEG-modified PEI greatly enhanced the transfection activity of the corresponding DNA complexes over the polyplexes containing PEG-modified PEI. The enhancements were observed at all N/P ratios tested and could be blocked partially by co-incubation with 200 microM free folic acid, which suggested the involvement of folate receptor in gene transfer. Targeted vectors based on the folate-PEG-PEI conjugate are potentially useful as simple tumor-specific vehicles of therapeutic genes.