RTL (RNase three-like) proteins belong to a distinct family of endonucleases that cleave double-stranded RNAs in plants. RTL1 to 3 are structurally related to the RNAse III from E. coli and formally belong to the class 1 of RNase III proteins. RTLs have conserved RNase III signature motif(s) and up to two dsRNA binding (DRB) domains. RTLs target and cleave coding and noncoding dsRNAs, including precursors of ribosomal (rRNA), small interference (siRNA), and micro (miRNA) RNAs. Interestingly, RTL proteins have stronger affinity than RNase III-Dicer proteins for dsRNA precursors of siRNAs, but not for miRNAs. However, very little is known of the structural and molecular bases directing and controlling RTL-RNA binding and activity. To address these questions, we have developed in vitro cleavage assays that combine recombinant RTL1 protein and in vitro transcribed or plant-extracted RNAs, RT-PCR, and primer extension experiments or analysis.