Freshwater planarian flatworms possess uncanny regenerative capacities mediated by abundant and collectively totipotent adult stem cells. Key functions of these cells during regeneration and tissue homeostasis have been shown to depend on PIWI, a molecule required for Piwi-interacting RNA (piRNA) expression in planarians. Nevertheless, the full complement of piRNAs and microRNAs (miRNAs) in this organism has yet to be defined. Here we report on the large-scale cloning and sequencing of small RNAs from the planarian Schmidtea mediterranea, yielding altogether millions of sequenced, unique small RNAs. We show that piRNAs are in part organized in genomic clusters and that they share characteristic features with mammalian and fly piRNAs. We further identify 61 novel miRNA genes and thus double the number of known planarian miRNAs. Sequencing, as well as quantitative PCR of small RNAs, uncovered 10 miRNAs enriched in planarian stem cells. These miRNAs are downregulated in animals in which stem cells have been abrogated by irradiation, and thus constitute miRNAs likely associated with specific stem-cell functions. Altogether, we present the first comprehensive small RNA analysis in animals belonging to the third animal superphylum, the Lophotrochozoa, and single out a number of miRNAs that may function in regeneration. Several of these miRNAs are deeply conserved in animals.microRNAs ͉ miRNAs ͉ piRNAs ͉ regeneration ͉ stem cells P lanarians have become a molecularly tractable model system in which to study regeneration, tissue homeostasis, and stem-cell biology (1). Planaria are free-living, triploblastic flatworms of the phylum Platyhelminthes, which is presently considered to belong to the superphylum Lophotrochozoa. Model systems for modern molecular and developmental biology have almost exclusively focused on the other 2 superphyla, i.e., the Deuterostomes (which includes vertebrates) and the Ecdysozoa (e.g., Caenorhabditis elegans and Drosophila melanogaster). Unlike these model systems, planarians possess remarkable regeneration abilities. Decapitation, for example, results in the complete regeneration of the head within 7 days after amputation. Such robust restoration of missing body parts is mediated by adult stem cells known as neoblasts (2). Of the thousands of known planarian species, Schmidtea mediterranea is arguably the species of choice for modern molecular biology and high-throughput, genome-wide approaches because it is diploid, it exists in sexual and asexual strains, and its genome has recently been sequenced and annotated (3). The size of its genome is roughly a third of the human genome, and Ϸ80% of the Ϸ20,000 annotated planarian genes have orthologs in humans. Moreover, by morphology alone, neoblasts and their immediate division progeny comprise Ϸ25% of all cells in the adult animal (4). In addition, RNAi screens have identified hundreds of genes specifically linked to planarian regeneration and stem-cell biology (5). Many of these genes are conserved in humans, and thus understanding planarian ...