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•Ser2 is highly phosphorylated in cardiac troponin T in vivo.•The sequence at Ser2 is conserved in cardiac troponin T of different species.•Ser2 is phosphorylated by troponin T kinase identical to casein kinase 2.•Phosphorylation of Ser2 does not affect the Ca-binding properties of troponin.•Ser2 phosphorylation is associated with still unknown long lasting processes.