RNA polymerase II carboxyl-terminal domain (pol II CTD) phosphatases that can dephosphorylate both Ser2-PO4 and Ser5-PO4 of CTD have been identified in animals and yeasts, however, only Ser5-PO4-specific CTD phosphatases have been identified in plants. Among predicted Arabidopsis SCP1-like small phosphatases (SSP), SSP4, SSP4b, and SSP5 form a unique group with long N-terminal extensions. While SSPs' expression showed similar tissue-specificities, SSP4 and SSP4b were localized exclusively in the nuclei, whereas SSP5 accumulated in both nuclei and cytoplasm. Detailed characterization of SSP activities using various peptides and full-length Arabidopsis pol II CTD substrates established that SSP4 and SSP4b could dephosphorylate both Ser2-PO4 and Ser5-PO4 of CTD, whereas SSP5 dephosphorylated only Ser5-PO4. These results indicate that Arabidopsis SSP gene family encodes active CTD phosphatases like animal SCP1 family proteins, with distinct substrate specificities. (C) 2010 Elsevier Inc. All rights reserved.