Deep sequencing of viral or bacterial nucleic acids monitors the presence and diversity of microbes in select populations and locations. Metagenomic study of mammalian viromes can help trace paths of viral transmissions within or between species. High throughput sequencing of patient and untreated sewage microbiomes showed many sequences with no similarity to genomic sequences of known function or origin. To estimate the distribution of functional RNAs in these microbiomes, we used the hammerhead ribozyme (HHR) motif to search for sequences capable of assuming its three-way junction fold. Although only two of the three possible natural HHR topologies had been known, our analysis revealed highly active ribozymes that terminated in any of the three stems. The most abundant of these are type II HHRs, one of which is the fastest natural cis-acting HHR yet discovered. Altogether, 13 ribozymes were confirmed in vitro, but only one showed sequence similarity to previously described HHRs. Sequences surrounding the ribozymes do not generally show similarity to known genes, except in one case, where a ribozyme is immediately preceded by a bacterial RadC gene. We demonstrate that a structure-based search for a known functional RNA is a powerful tool for analysis of metagenomic datasets, complementing sequence alignments.RNAs fulfill diverse biological roles, including regulation and catalysis. The discovery of catalytic RNAs over the last 30 years supports the RNA world hypothesis, which proposes that RNA predated proteins as the information carrier and the catalytic macromolecule (1). Biological catalytic RNAs include phosphotransferases and the ribosomal peptidyl transferase (2, 3). Phosphotransferases include six types of naturally occurring nucleolytic ribozymes capable of cleaving phosphodiester linkages: hammerhead (HHR) 2 (4), hairpin (5), hepatitis delta virus (HDV) (6, 7), the Neurospora Varkud satellite (8), the bacterial cofactor-dependent GlmS (9), and in some cases, group I intron-like ribozymes (10, 11). Early work identified these selfcleaving RNAs through analysis of single gene transcripts and pathogen genomes (12). In addition, in vitro selection experiments have identified a variety of self-cleaving ribozymes (13), including the hammerhead motif, which was found independently several times (14). In recent years, structure-based searches and in vitro selection from a genomic library have revealed hammerhead and HDV-like ribozymes in many species ranging from bacteria to mammals (15)(16)(17)(18)(19)(20).Hammerhead ribozymes were originally discovered in plant viroids and virusoids, where they function in the processing of rolling circle transcripts (21). HHRs are also known to exist in the satellite transcripts of various newt species (22), cave crickets (23), and the human blood fluke Schistosoma mansoni (24). The recent discoveries of HHRs in many different species, including mammals, suggest multiple biological functions (15,19,20).The HHR structure consists of three helices (stems I, II, and III) ancho...