Previously we have shown that in vitro-packaged simian virus 40 (SV40) pseudovirions (IVPs) are an efficient delivery system for supercoiled DNA plasmids of up to 17.7 kb, with or without SV40 sequences. RNA interference (RNAi) is a naturally occurring gene-silencing mechanism mediated by small double-stranded RNA molecules (small interfering RNAs, siRNAs). This study demonstrates the first use of SV40 pseudovirions to deliver into human cells both principal types of RNAi effector molecules: plasmid-expressed short hairpin RNAs (shRNAs) and synthetic siRNAs. We first established the ability of human lymphoblastoid cells to support RNAi, using sequential transduction of .45 cells with packaged plasmid DNA expressing the green fluorescent protein (IVP-GFP), and an shRNA corresponding to the GFP (IVP-shGFP). SV40 mediates DNA transfer of nucleic acid to the cytoplasm, where RNAi-associated cleavage of mRNA principally occurs. Using SV40 pseudovirions, siRNA-mediated RNAi was observed in both .45 cells, after sequential transduction of IVP-GFP and IVP-packaged siRNAs corresponding to GFP (IVP-siGFP), and in HeLa cells stably expressing a GFP transduced with IVP-siGFP. Our findings indicate that SV40 pseudovirions may be a useful addition to the delivery systems currently being used for the transfer of RNAi effector molecules.