Tet proteins can convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). This study shows that Tet proteins, in addition to converting 5mC to 5hmC, can also generate 5fC and 5caC through enzymatic activity. These modified forms of cytosine were detected in genomic DNA of mouse embryonic stem (ES) cells and organs. The levels of 5hmC, 5fC, and 5caC can be altered by overexpression or depletion of Tet proteins, indicating that these are products of Tet activity. The study suggests that DNA demethylation may occur through Tet-catalyzed oxidation followed by decarboxylation. The findings indicate that Tet proteins not only convert 5mC to 5hmC but also further oxidize 5hmC to 5fC and 5caC. The presence of 5fC and 5caC in genomic DNA was confirmed using 2D-TLC and mass spectrometry. The study also shows that Tet proteins can act on 5hmC and 5fC-containing substrates. The kinetic analysis of Tet2 revealed that it can convert 5mC to 5hmC, 5fC, and 5caC. The presence of 5fC and 5caC in genomic DNA of mouse ES cells and organs was confirmed. The study also shows that the abundance of these modified cytosines is regulated by Tet proteins. The results suggest that Tet-catalyzed oxidation may be a key pathway for DNA demethylation. The study provides evidence that 5fC and 5caC are present in genomic DNA under physiological conditions. The findings highlight the role of Tet proteins in DNA demethylation and the potential for further research into the mechanisms of DNA modification.Tet proteins can convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC). This study shows that Tet proteins, in addition to converting 5mC to 5hmC, can also generate 5fC and 5caC through enzymatic activity. These modified forms of cytosine were detected in genomic DNA of mouse embryonic stem (ES) cells and organs. The levels of 5hmC, 5fC, and 5caC can be altered by overexpression or depletion of Tet proteins, indicating that these are products of Tet activity. The study suggests that DNA demethylation may occur through Tet-catalyzed oxidation followed by decarboxylation. The findings indicate that Tet proteins not only convert 5mC to 5hmC but also further oxidize 5hmC to 5fC and 5caC. The presence of 5fC and 5caC in genomic DNA was confirmed using 2D-TLC and mass spectrometry. The study also shows that Tet proteins can act on 5hmC and 5fC-containing substrates. The kinetic analysis of Tet2 revealed that it can convert 5mC to 5hmC, 5fC, and 5caC. The presence of 5fC and 5caC in genomic DNA of mouse ES cells and organs was confirmed. The study also shows that the abundance of these modified cytosines is regulated by Tet proteins. The results suggest that Tet-catalyzed oxidation may be a key pathway for DNA demethylation. The study provides evidence that 5fC and 5caC are present in genomic DNA under physiological conditions. The findings highlight the role of Tet proteins in DNA demethylation and the potential for further research into the mechanisms of DNA modification.