Posttranscriptional suppression of gene expression can be achieved by introduction of sequence-specific small interfering (si) RNA duplexes and by de novo intracellular synthesis of short sequence-specific double-stranded RNAs. However, achieving desired levels of knockdown is a barrier to successful analytic and therapeutic application. We demonstrate that increasing expression of introduced short hairpin RNA (shRNA) can markedly enhance RNA interference (RNAi) and that this approach can be used to achieve maximal target down-regulation, when the choice of optimal siRNA-binding sites is restricted or when multiple genes are simultaneously targeted and the amount of siRNA is limiting. A dose-dependent RNAi effect was accomplished by placing copies of shRNA under control of the Pol III U6 small nuclear RNA promoter in tandem in a DNA vector. Using this system, we achieved simultaneous down-regulation of expression of classical human leukocyte antigen (HLA) class I genes in cultured and primary human T cells, which might be applied to help circumvent T-cell-mediated rejection of immunogenic and/or HLA-disparate allografts.