Exploiting the phenomenon of RNA interference (RNAi), recent studies established the utility of monospecific small interfering RNAs (siRNAs) in suppressing HIV-1 infection. However, because of the high mutation rate of the HIV genome, there are considerable challenges in the design of fully efficacious gene therapeutic constructs. Therefore, approaches that simultaneously target different stages of the viral life cycle are desirable. In our current studies, we designed bispecific siRNA constructs against HIV-1 cell surface receptors to inhibit viral entry. Dual specific short hairpin siRNA constructs, containing an 8-nucleotide intervening spacer, targeted against either CXCR4 and CD4 or CCR5 and CXCR4 were synthesized by in vitro transcription. Cleavage of the bispecific constructs yielding monospecific siRNAs was shown to occur in cell extracts. Magi-CXCR4 and CCR5 cells transfected with bispecific siRNAs showed significant downregulation of their respective coreceptors, as determined by FACS analysis. This suggested that combinatorial constructs comprising multiple effector motifs were processed in transfected cells into their respective functional siRNAs. Transfected cells were challenged with either X4 (NL4-3) or R5-tropic (BaL-1) strains of HIV-1. Downregulation of the cell surface receptors coincided with resistance to in vitro viral challenge in both Magi cell lines and peripheral blood mononuclear cells (PBMCs). These results demonstrated the practical utility of short hairpin siRNA bispecific constructs synthesized as a single transcript. Because the short hairpin design will permit tandem assembly of multiple effector motifs, it is now possible to introduce promising multivalent siRNA constructs into retroviral and lentiviral vectors for in vivo gene therapeutic applications.