PIWI-interacting RNAs (piRNAs) are single-stranded, 23–36 nucleotide (nt) RNAs that act as guides for an animal-specific class of Argonaute proteins, the PIWI proteins. The first piRNAs — derived from the Suppressor of Stellate locus in Drosophila melanogaster testes — were discovered in 2001. Although the authors noted the larger size of those ‘rasiRNAs’ (repeat-associated siRNAs), piRNAs were not recognized until 2006 as a distinct class of small silencing RNAs that derive from single-stranded, rather than double-stranded RNA (dsRNA), precursors. It was their association with PIWI, but not AGO, proteins and their independence from Dicer — the RNase III enzyme that cleaves dsRNAs to release small interfering RNAs (siRNAs) — that finally distinguished piRNAs from siRNAs. Moreover, while siRNAs are expressed ubiquitously, piRNAs are predominantly found in animal gonads and are thought to be indispensable for fertility.