Three novel genes encoding small RNAs homologous to human and mouse RNase P RNA have been isolated from a mouse genomic library. As assessed by Northern blot analysis and nuclease protection assays, transcripts derived from one or more of these genes are expressed in murine cells and tissues. The RNA products of these RNase P RNA-homologous genes are smaller in size (238-248 nucleotides) than the 305-nucleotide transcript previously identified. These smaller transcripts are uniformly less abundant than the larger RNase P RNA, but their expression varies severalfold among different mouse tissues. Similar short homologues of RNase P RNA also are expressed in rat, rabbit, and human cells. We conclude that higher vertebrates express multiple isoforms of RNase P RNA.