The term nanodisk (ND) describes reconstituted high-density lipoprotein particles that contain one or more exogenous bioactive agents. In the present study, ND were assembled from apolipoprotein A-I, the zwitterionic glycerophospholipid 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), and the synthetic cationic lipid 1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP). ND formulated at a DMPC:DMTAP ratio of 70:30 (by weight) were soluble in aqueous media. The particles generated were polydisperse, with diameters ranging from ∼20 to <50 nm. In nucleic acid binding studies, agarose gel retardation assays revealed that a synthetic 23-mer double-stranded oligonucleotide (dsOligo) bound to DMTAP containing ND but not to ND formulated with DMPC alone. Sucrose density gradient ultracentrifugation studies provided additional evidence for stable dsOligo binding to DMTAP–ND. Incubation of cultured hepatoma cells with DMTAP–ND complexed with a siRNA directed against glyceraldehyde 3-phosphate dehydrogenase showed 60% knockdown efficiency. Thus, incorporation of synthetic cationic lipid (i.e., DMTAP) to ND confers an ability to bind siRNA and the resulting complexes possess target gene knockdown activity in a cultured cell model.