Xenopus laevis is an excellent model for thyroid hormone (T3)-regulated gene expression. T3 initiates two drastically different pathways during metamorphosis: death of larval tissues and growth of adult tissues. The role that each T3 receptor (TR) isotype, alpha and beta, plays in metamorphosis is uncertain. The X.laevis tetraploid genome limits experiments to overexpression, misexpression and dominant negative studies. Ribozymes offer an alternative by suppressing gene activity through specific mRNA reduction. It has been suggested that ribozymes will not work in X.laevis because of the organism's intracellular environment and body temperature. In this study, we show that hammerhead ribozymes are active in vitro against transcribed TRbeta message and in vivo against a TRbeta-luciferase fusion protein. We next show that TRbeta-targeted ribozymes can inhibit T3-induced transcription of a reporter gene in cultured X.laevis cells, using T3 response elements from two T3-responsive transcription factor genes. One has early expression kinetics in response to T3 and is proposed to be TRalpha regulated whereas the other has intermediate induction kinetics and thus may be partially TRbeta regulated. Therefore, ribozymes are a potentially valuable tool for overcoming the limitations in this system for examining gene function in X.laevis.