PIWI proteins and PIWI-interacting RNAs (piRNAs) mediate repression of transposons in the animal gonads. Primary processing converts long single-stranded RNAs into approximately 30-nt piRNAs, but their entry into the biogenesis pathway is unknown. Here, we demonstrate that an RNA element at the 5' end of a piRNA cluster-which we termed piRNA trigger sequence (PTS)-can induce primary processing of any downstream sequence. We propose that such signals are triggers for the generation of the original pool of piRNAs. We also demonstrate that endonucleolytic cleavage of a transcript by a cytosolic PIWI results in its entry into primary processing, which triggers the generation of non-overlapping, contiguous primary piRNAs in the 3' direction from the target transcript. These piRNAs are loaded into a nuclear PIWI, thereby linking cytoplasmic post-transcriptional silencing to nuclear transcriptional repression.