Endonucleases are key effectors of mRNA degradation, particularly for mRNAs whose turnover rates are regulated by extracellular stimuli. The rapid clearance of mRNA degradation products in vivo and the need to selectively identify mRNA endonucleases in the presence of many other cellular ribonucleases make the study of these enzymes particularly challenging. We have successfully purified and cloned one such enzyme, termed polysomal RNase 1, or PMR-1. Presented here are protocols either developed in our laboratory or adapted from the work of others that we have used successfully in characterizing PMR-1. We first describe methods to determine whether a particular mRNA is degraded in vivo through an endonuclease-initiated mechanism, and then present approaches for developing an in vitro mRNA degradation system. Next we describe experiments one should perform to optimize reaction conditions, determine cofactor requirements for an endonuclease, map in vitro cleavage sites, and characterize endonucleolytic cleavage products. Finally we describe kinetic parameters one should evaluate in characterizing the enzymology of mRNA endonucleases, with particular concern focused on the relative selectivity of these enzymes for cleavage at preferred sites within target mRNAs.