Hammerhead ribozymes are catalytic RNA molecules that can act in trans, with ribozyme and substrate being two different oligoribonucleotides with regions of complementarity. Mutations in the gene for fibrillin-1 (FBN1) cause Marfan syndrome. The majority of mutations are single-base changes, many of which exert their effect via a dominant-negative mechanism. Previously we have shown that an antisense hammerhead ribozyme, targeted to the FBN1 mRNA can reduce deposition of fibrillin to the extracellular matrix of cultured fibroblasts, suggesting it may be possible to utilize ribozymes to down regulate the production of mutant protein and thus restore normal fibrillin function. This might be achieved by the mutation creating a ribozyme cleavage site that is not present in the normal allele, however this is likely to limit the number of mutations that could be targeted. Alternatively, it might be possible to target the mutant allele via the ribozyme binding arms. To determine the potential of ribozymes to preferentially target mutant FBN1 alleles via the latter approach, the effect of mismatches in helix I of a hammerhead ribozyme, on the cleavage of fibrillin (FBN1) mRNA was investigated. A single base mismatch significantly reduced ribozyme cleavage efficiency both in vitro and in vivo. The discrimination between fully-matched and mismatched ribozyme varied with the length of helix I, with the discrimination being more pronounced in ribozymes with a shorter helix. These data suggest that it should be possible to design hammerhead ribozymes that can discriminate between closely related (mutant and normal) target RNAs varying in as little as a single nucleotide, even if the mutation does not create a ribozyme cleavage site.