Dicer proteins are known to produce small RNAs (sRNAs) from long double-stranded RNA (dsRNA) templates. These sRNAs are bound by Argonaute proteins， which select the guide strand， often with a 5' end sequence bias. However， Dicer proteins have never been shown to have sequence cleavage preferences. In Paramecium development， two classes of sRNAs that are required for DNA elimination are produced by three Dicer-like enzymes: Dcl2， Dcl3， and Dcl5. Through in?vitro cleavage assays， we demonstrate that Dcl2 has a strict size preference for 25 nt and a sequence preference for 5' U and?5'?AGA， while Dcl3 has a sequence preference for 5'?UNG. Dcl5， however， has cleavage preferences?for 5' UAG and 3' CUAC/UN， which leads to the production of RNAs precisely matching short excised DNA elements with corresponding end base preferences. Thus， we characterize three Dicer-like enzymes that are involved in Paramecium development and propose a biological role for their sequence-biased cleavage products.