In eukaryotes, the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) is composed of tandem repeats of the heptapeptide YSPTSPS, which is subjected to reversible phosphorylation at Ser2, Ser5, and Ser7 during the transcription cycle. Dynamic changes in CTD phosphorylation patterns, established by the activities of multiple kinases and phosphatases, are responsible for stage-specific recruitment of various factors involved in RNA processing, histone modification, and transcription elongation/termination. Yeast Ssu72, a CTD phosphatase specific for Ser5 and Ser7, functions in 3'-end processing of pre-mRNAs and in transcription termination of small non-coding RNAs such as snoRNAs and snRNAs. Vertebrate Ssu72 exhibits Ser5- and Ser7-specific CTD phosphatase activity in vitro, but its roles in gene expression and CTD dephosphorylation in vivo remain to be elucidated. To investigate the functions of vertebrate Ssu72 in gene expression, we established chicken DT40 B-cell lines in which Ssu72 expression was conditionally inactivated. Ssu72 depletion in DT40 cells caused defects in 3'-end formation of U2 and U4 snRNAs and GAPDH mRNA. Surprisingly, however, Ssu72 inactivation increased the efficiency of 3'-end formation of non-polyadenylated replication-dependent histone mRNA. Chromatin immunoprecipitation analyses revealed that Ssu72 depletion caused a significant increase in both Ser5 and Ser7 phosphorylation of the Pol II CTD on all genes in which 3'-end formation was affected. These results suggest that vertebrate Ssu72 plays positive roles in 3'-end formation of snRNAs and polyadenylated mRNAs, but negative roles in 3'-end formation of histone mRNAs, through dephosphorylation of both Ser5 and Ser7 of the CTD.