gamma-Glutamylcysteine synthetase (gamma-GCS) is a key enzyme in glutathione synthesis and is thought to play a significant role in intracellular detoxification systems. To specifically suppress gamma-GCS gene expression, we constructed two different hammerhead ribozymes against gamma-GCS mRNA transcripts. Two cleavage sites were targeted as follows: site 1 for anti-gamma-GCS ribozyme (H), a GUU triplet located from +348 to +350 of the gamma-GCS heavy chain, and site 2 for anti-gamma-GCS ribozyme (L), a GUU triplet located from +235 to +237 of the gamma-GCS light chain. The anti-gamma-GCS ribozymes effectively cleaved gamma-GCS mRNA in a cell-free system. When transfected into a Min-6 mouse islet cell line, these anti-gamma-GCS ribozymes not only suppressed gamma-GCS gene expression, but also reduced intracellular glutathione concentration. These results suggest that the ribozyme-mediated down-regulation of gamma-GCS gene expression may be useful for analyzing the glutathione-associated cellular defense systems of pancreatic islet cells.