Vaults are large cytoplasmic ribonucleoprotein particles with a sedimentation value of about 150 S. These particles contain a unique small RNA (vault RNA (vRNA)). We have determined the sequence of the RNA associated with vaults purified from both rat and bullfrog. The rat vRNA is 141 bases in length, whereas the bullfrog vRNA is present as two highly related species of 89 and 94 bases. Despite the differences in length the predicted secondary structures of the three vRNAs are clearly related. All of the vRNAs contain sequences related to the internal promoter elements necessary for transcription by RNA polymerase III. The gene for the rat vRNA was isolated and sequenced from a rat genomic library, and its transcription by RNA polymerase III was verified using an in vitro transcription assay. The rat vRNA gene was efficiently transcribed in vitro, producing a single transcript of about 140 bases. Unlike most RNA polymerase III genes, the rat vRNA is present as a single copy gene and has a distinct tissue-specific expression pattern.