Mitochondria import from the cytoplasm the vast majority of proteins and some RNAs. Although there exists extended knowledge concerning the mechanisms of protein import, the import of RNA is poorly understood. It was almost exclusively studied on the model of tRNA import, in several protozoans, plants and yeast. Mammalian mitochondria, which do not import tRNAs naturally, are hypothesized to import other small RNA molecules from the cytoplasm. We studied tRNA import in the yeast system, both in vitro and in vivo, and applied similar approaches to study 5S rRNA import into human mitochondria. Despite the obvious divergence of RNA import systems suggested for different species, we find that in yeast and human cells this pathway involves similar mechanisms exploiting cytosolic proteins to target the RNA to the organelle and requiring the integrity of pre-protein import apparatus. The import pathway might be of interest from a biomedical point of view, to target into mitochondria RNAs that could suppress pathological mutations in mitochondrial DNA. Yeast represents a good model to elaborate such a gene therapy approach. We have described here the various approaches and protocols to study RNA import into mitochondria of yeast and human cells in vitro and in vivo.