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Small RNAs ranging in size between 20 and 30 nucleotides are involved in different types of regulation of gene expression including mRNA degradation, translational repression, and chromatin modification. Here we describe the small RNA profile of Drosophila melanogaster as a function of development. We have cloned and sequenced over 4000 small RNAs, 560 of which have the characteristics of RNase III cleavage products. A nonredundant set of 62 miRNAs was identified. We also isolated 178 repeat-associated small interfering RNAs (rasiRNAs), which are cognate to transposable elements, satellite and microsatellite DNA, and Suppressor of Stellate repeats, suggesting that small RNAs participate in defining chromatin structure. rasiRNAs are most abundant in testes and early embryos, where regulation of transposon activity is critical and dramatic changes in heterochromatin structure occur.