Identification of sequences within a target mRNA that are susceptible to potent siRNA knockdown often requires testing several independent siRNAs or shRNA expression cassettes. Using RNAi against HIV RNAs is further complicated by the length of the viral genome, the complexity of splicing patterns, and the propensity for genetic heterogeneity; consequently, it is most important to identify a number of siRNA targets that potently block viral replication. We previously described a facile PCR-based strategy for rapid synthesis of si/shRNA expression units and their testing in mammalian cells. Using this approach, which is rapid and inexpensive, it is possible to screen a number of potential RNAi targets in HIV to identify those that are most susceptible to RNAi. We report that shRNA expression cassettes constructed by PCR and cotransfected directly into mammalian cells with HIV proviral DNA express shRNAs that are inhibitory to HIV-1 replication. Our results also demonstrate that there is a wide range of efficacies among shRNAs targeting different sites throughout the HIV genome. By screening several different targets we were able to identify a sequence in a common tat/rev exon that is exquisitely sensitive to RNAi. Furthermore we relate the efficacies of our PCR product expressed shRNAs to the relative stabilities of the siRNA duplexes and the accessibilities of the target sites to antisense base pairing in cell extracts.