Genetic transformation of mitochondria in multicellular eukaryotes has remained inaccessible, hindering fundamental investigations and applications to gene therapy or biotechnology. In this context, we have developed a strategy to target nuclear transgene-encoded RNAs into mitochondria in plants. We describe here mitochondrial targeting of trans-cleaving ribozymes destined to knockdown organelle RNAs for regulation studies and inverse genetics and biotechnological purposes. The design and functional assessment of chimeric RNAs combining the ribozyme and the mitochondrial shuttle are detailed, followed by all procedures to prepare constructs for in vivo expression, generate stable plant transformants, and establish target RNA knockdown in mitochondria.