Background: RNA interference (RNAi) was originally identified as a biological process in which short double-stranded RNA (dsRNA) suppress the expression of genes complimentary to the dsRNA. This cellular intrinsic gene silencing mechanism has subsequently been developed as a useful tool for studies of gene function. A major strategy for producing small interfering RNA (siRNA) in cultured cells involves the use of siRNA expression vectors in which a RNA polymerase III (Pol III) promoter and transcription stop signal are designed to constitute a functional siRNA expression cassette for production of siRNA. However, most of the available vectors contain only one siRNA expression cassette. Results: In order to maximize the efficiency and versatility of the vector-based siRNA approach, we have developed vectors containing multiple (up to six) tandem siRNA expression cassettes. Moreover, we demonstrated that these vectors can be used not only to produce different siRNA to simultaneously suppress the expression of multiple genes but also to maximize the silencing of a singe gene. Conclusion: The vectors containing multiple siRNA expression cassettes can serve as useful tools for maximizing the efficiency of gene silencing. © 2006 Wang et al; licensee BioMed Central Ltd.