HIV-1 infection of susceptible cells is mediated by the specific interaction of viral envelope glycoproteins with the cell surface CD4 receptor and a chemokine coreceptor, CCR5 or CXCR4. Individuals with a CCR5 genetic defect show resistance to HIV-1 infection, indicating that downregulation of CCR5 expression on target cells can prevent viral infection. In previous studies we demonstrated the utility of an anti-CCR5 ribozyme targeted to a single cleavage site in downregulating CCR5 expression and consequently providing resistance to viral infection. To improve on the level of downregulation we designed a construct containing an anti-CCR5 ribozyme heterotrimer (R5RbzTM) targeted to three different cleavage sites in CCR5 mRNA. In vitro tests showed that the anti-CCR5 ribozyme heterotrimer could effectively cleave the CCR5 RNA substrates to yield products of the expected sizes. This construct was introduced into various retroviral vectors for stable gene transduction. HOS.CD4/R5 cells stably transduced with this anti-CCR5 heterotrimer showed a marked reduction in the surface expression of CCR5 and a concomitant 70% reduction in macrophage-tropic viral infection. In addition, a retroviral vector containing the anti-CCR5 ribozyme heterotrimer and an anti-HIV-1 tat-rev ribozyme heterodimer was constructed. This construct also showed a similar inhibition of CCR5 surface expression and reduced infectability by the macrophage-tropic HIV-1 vector in HOS.CD4/R5 cells. The trimeric and multimeric ribozyme constructs were transduced into CD34+ hematopoietic progenitor cells to determine their effects on lineage-specific differentiation. We show that multivalent ribozyme gene-transduced hematopoietic progenitors differentiated normally into mature macrophages that bear CD14 and CD4 surface markers. Macrophages containing the transgenes expressed ribozymes, and showed resistance to M-tropic HIV-1 infection. These results provide strong support for the use of the trimeric anti-CCR5 ribozyme approach in a gene therapy setting for the treatment of HIV infection.