We have previously described a hairpin ribozyme that targets a highly conserved sequence in the U5 region of HIV-1. To determine if escape mutations would compromise virus replication, we introduced critical mutations into the ribozyme target site of an infectious molecular clone of HIV-1MN. HIV-1 MNA has a substitution of A for G immediately 3' to the cleavage site and HIV-1 MNGC has two substitutions in the flanking sequences that are complementary to the ribozyme. In vitro studies confirmed that neither the MNA-nor the MNGC-mutated target sequence was cleaved by the ribozyme, and furthermore, the MNGC-mutated target sequence failed to bind the ribozyme. Compensatory GC substitutions in the substrate recognition domain of the ribozyme resulted in a switch of binding and cleavage specificity. Replication of both the MNA and MNGC mutant viruses was initially two to three logs lower than that of wild-type virus, but after 3 weeks, virus production rose sharply in both cultures. Nucleotide sequence of RT-PCR-amplified viral sequences obtained from virus produced at later time points revealed complete reversion of MNA or partial reversion of MNGC to wild-type genotypes. No additional mutations within the ribozyme target sequence were observed. These results indicate that mutations in this conserved ribozyme target sequence led to significant attenuation of HIV-1MN.