This study investigates the three isomers of lysine lactylation (K_L-la, K_D-la, and K_ce) and their relationship with glycolysis. The researchers developed methods to distinguish these isomers, including chemical derivatization, high-performance liquid chromatography (HPLC), and isomer-specific antibodies. They found that K_L-la is the dominant lactylation isomer on histones and is dynamically regulated by glycolysis, unlike K_D-la and K_ce, which are observed when the glyoxalase system is incomplete. The study also reveals that lactyl-coenzyme A, a precursor in L-lactylation, correlates positively with K_L-la levels. The findings highlight K_L-la as the primary responder to glycolysis and the Warburg effect. The research provides a methodology for distinguishing other PTM isomers and underscores the importance of K_L-la in cellular metabolism and epigenetic regulation. The study also demonstrates that K_L-la is more prevalent on histones than K_D-la or K_ce, and that it is responsive to changes in glycolysis. The results suggest that K_L-la is primarily a modification catalyzed by acyltransferases with lactyl-CoA as a cofactor, while K_D-la and K_ce are produced by nonenzymatic reactions. The study concludes that K_L-la is the dominant isomer of lysine lactylation on cellular histones and is regulated by glycolysis.This study investigates the three isomers of lysine lactylation (K_L-la, K_D-la, and K_ce) and their relationship with glycolysis. The researchers developed methods to distinguish these isomers, including chemical derivatization, high-performance liquid chromatography (HPLC), and isomer-specific antibodies. They found that K_L-la is the dominant lactylation isomer on histones and is dynamically regulated by glycolysis, unlike K_D-la and K_ce, which are observed when the glyoxalase system is incomplete. The study also reveals that lactyl-coenzyme A, a precursor in L-lactylation, correlates positively with K_L-la levels. The findings highlight K_L-la as the primary responder to glycolysis and the Warburg effect. The research provides a methodology for distinguishing other PTM isomers and underscores the importance of K_L-la in cellular metabolism and epigenetic regulation. The study also demonstrates that K_L-la is more prevalent on histones than K_D-la or K_ce, and that it is responsive to changes in glycolysis. The results suggest that K_L-la is primarily a modification catalyzed by acyltransferases with lactyl-CoA as a cofactor, while K_D-la and K_ce are produced by nonenzymatic reactions. The study concludes that K_L-la is the dominant isomer of lysine lactylation on cellular histones and is regulated by glycolysis.