Plastid casein kinase II is an important regulator of transcription, posttranscriptional processes, and, most likely, different metabolic functions in dicotyledonous species. Here we report the identificationand characterization of pCKII from the monocotyledonous species Oryza sativa. OspCKII activity was enriched from isolated ricechloroplasts using heparin-Sepharose chromatography, in which it co-elutes with the transcriptionally active chromosome (TAC) and several ribosomal proteins. Inclusion mass scanning of the kinase-active fraction identified the gene model for OspCKII. Transient expression of GFP fused to the 184 N-terminal amino acids of the OspCKII sequence in rice confirmed the chloroplastic localization of the kinase. OspCKII activity shows the characteristic features of casein kinase II, such as the utilization of GTP as phosphate donor, inhibition by low concentrations ofheparin and poly-lysine, and utilization of the canonical pCKII motif E-S-E-G-E in the model substrate RNP29. Phosphoproteome analysis of a protein extract from rice leaves combined with a meta-analysis with published phosphoproteomics data revealed differences in the target protein spectrum between rice and Arabidopsis. Consistently, several pCKII phosphorylation sites in dicotyledonous plants are not conserved in monocots and algae, suggesting that details of pCKII regulation in plastids have changed during evolution.