The biogenesis of animal microRNAs (miRNAs) involves transcription followed by a series of processing steps, with Drosha and Dicer being two key enzymes that cleave primary miRNA (pri-miRNA) and precursor miRNA (pre-miRNA) transcripts, respectively. While human and fly Dicer and human Drosha are well studied, their homologs in other organisms have not been biochemically characterized, leaving open the question of whether their miRNA substrate specificities and regulatory functions are conserved. Zebrafish is a widely used model organism, but its miRNA processing enzymes have never been reconstituted and analyzed. In this study we cloned and constructed expression plasmids encoding zebrafish Dicer, Drosha, and their accessory proteins TARBP2 and DGCR8. After transfection of human cell cultures, we isolated the recombinant protein complexes. We found that zebrafish Dicer bound TARBP2, but Dicer alone exhibited significant pre-miRNA processing activity. On the other hand, zebrafish Drosha associated with DGCR8, and both were required to cleave pri-miRNAs. The Drosha/DGCR8 holoenzyme preferred pri-miRNAs with a large terminal loop, an extended duplex region, and flanking single stranded RNAs. These results lay the foundation for future studies of the regulatory roles and conserved mechanisms of Drosha and Dicer. (C) 2019 Elsevier Inc. All rights reserved.