AbstractEndonuclease‐mediated mRNA decay appears to be a common mode of mRNA degradation in mammalian cells, but yet only a few mRNA endonucleases have been described. Here, we report the existence of a second mammalian endonuclease that is capable of cleaving c‐myc mRNA within the coding region in vitro. This study describes the partial purification and biochemical characterization of this enzyme. Five major proteins of ∼10–35 kDa size co‐purified with the endonuclease activity, a finding supported by gel filtration and glycerol gradient centrifugation analysis. The enzyme is an RNA‐specific endonuclease that degrades single‐stranded RNA, but not double‐stranded RNA, DNA or DNA–RNA duplexes. It preferentially cleaves RNA in between the pyrimidine and purine dinucleotides UA, UG, and CA, at the coding region determinant (CRD) of c‐myc RNA. The enzyme generates products with a 3′hydroxyl group, and it appears to be a protein‐only endonuclease. It does not possess RNase A‐like activity. The enzyme is capable of cleaving RNAs other than c‐myc CRD RNA in vitro. It is Mg2+‐independent and is resistant to EDTA. The endonuclease is inactivated at and above 70°C. These properties distinguished the enzyme from other previously described vertebrate endonucleases. J. Cell. Biochem. 98: 519–537, 2006. © 2005 Wiley‐Liss, Inc.