The cleavage of target mRNA by ribozymes is being exploited as a means of gene silencing in nucleic-acidbased therapies. We previously established an HIV-1-dependent ribozyme-expression vector system, based on Cre-loxP technology with an LTR-gag-p17 promoter as a molecular switch for use in acute HIV-1 infection. The simultaneous expression of the Cre protein and loxP homologous recombination induced a high level of HIV-1-replication inhibition, but ribozyme expression was transient. In the current study, we overcame this limitation by inserting EBNA-1 and oriP genes from the Epstein-Barr virus (EBV) into the vector. When this plasmid was introduced into HeLa CD4+ cells, we observed long-term expression of both the EGFP reporter gene and the ribozyme. Moreover, HIV-1 replication was inhibited in the long-term in transfected cells. These data suggest that the HIV-1-dependent ribozyme-expression vector containing EBNA-1/oriP sequences would be a useful tool in HIV-1 gene therapy applications.