In different types of human cancer there is an overexpression of the c-erbB-2 (HER2/neu) oncogene, which is thought to be involved in tumor progression. Therefore the c-erbB-2 oncogene is an attractive target for tumor-specific gene therapy. In this report we have characterized a hammerhead ribozyme against the c-erbB-2 mRNA with high cleavage activity. To select the optimum sequence, the activity of five hammerhead ribozymes was tested in a cell-free assay. The hammerhead ribozyme recognizing the GUC sequence at position +631 to +633 of the c-erbB-2 mRNA (RZ631) efficiently cleaves in vitro transcribed fragments of the c-erbB-2 mRNA [169 to 1450 nucleotides (nt)] under multiple-turnover conditions. The ribozyme coding sequence was subsequently cloned between the A and the B box promoter sequences of the fowl adenovirus type 1 virus-associated RNA (CELO VA) gene. The in vitro activity of RZ631 was shown to be unaffected by the polymerase III promoter flanking sequences. The ability of RZ631 to inhibit the synthesis of the c-erbB-2 gene product in tumor cells was assayed by cotransfection of the ribozyme with a fusion gene of c-erbB-2 and the gene for the enhanced green fluorescent protein as a reporter. The synthesis of the fluorescent fusion protein in NIH:OVCAR-3 ovarian cancer cells was potently inhibited by RZ631, as assayed by flow cytometry. An antisense control vector, where the catalytic core was replaced by a single base, showed a weaker inhibition of expression of the c-erbB-2 derivative. The results suggest that the inhibitory effect of this c-erbB-2 ribozyme is caused by an antisense effect as well as by an additional ribozyme-mediated increase in inhibition. We conclude that this c-erbB-2 ribozyme in conjunction with a polymerase III-based expression system should be useful for the efficient downregulation of the c-erbB-2 oncogene in ovarian cancer cells.