Chloroplasts have a complex enzymatic machinery to adjust the relative half-life of their mRNAs to environmental signals. Soluble protein extracts from spinach (Spinacia oleracea L.) chloroplasts that correctly reproduce in vitro the differential mRNA stability observed in vivo were analyzed using shotgun proteomics to identify the proteins that are potentially involved in this process. The combination of a novel strategy for the database-independent detection of proteins from MS/MS data with standard database searches allowed us to identify 243 proteins with high confidence, which include several nucleases and RNA binding proteins but also proteins that have no reported function in chloroplast mRNA metabolism. Characterization of enzyme activities that adjust mRNA stability in response to illumination revealed that the dark-induced RNA degradation pathway involves enzymatic activities that differ from those that direct RNA processing and stabilization in the light. Dark-induced mRNA degradation comprises a MgCl2-independent and a MgCl2-dependent step, which releases nucleoside di- and monophosphates from the petD 3'-UTR precursor substrate. RNA degradation can be blocked with RNasin, a potent inhibitor of eukaryotic ribonucleases, suggesting that chloroplast mRNA degradation involves enzymes that are distinct from those found in prokaryotic-type RNA degradation. On the basis of the identified proteins and the in vitro characterization of the RNA degradation activities, we discuss scenarios and components that potentially determine plastid mRNA stability.