In budding yeast, there are five JmjC domain-containing proteins, Jhd1, Jhd2, Rph1, Ecm5, and Gis1, which have been suggested to directly remove histone lysine methylation via a hydroxylation reaction. Of these demethylases, the ability of Jhd1 or Rph1 to demethylate histone H3 as a substrate has been identified in vivo. However, the overall roles of endogenous JmjC demethylases in the demethylation of histones encompassed by genes that are constitutively transcribed or their specificities towards histone H3 lysine modification at mono-, di-, or trimethylation states are still unclear. Using chromatin immunoprecipitation with nine specific antibodies directed against mono-, di-, or trimethylated histone H3 at lysines 4, 36, or 79, we show the whole patterns of histone H3 lysine methylation and the net changes in methylations that are caused by the deletion of each of the five JmjC demethylases in actively transcribed regions. Our results show that of the JmjC-containing proteins, Rph1 is the demethylase that is specific for histone H3K36 trimethylation during transcription elongation in vivo, and the abilities of other endogenous JmjC demethylasesto demethylate histone H3 are weak toward histone H3in actively transcribed regions. (C) 2011 Elsevier Inc. All rights reserved.