Cardiac troponin I (cTnI), the inhibitory subunit of the thin filament troponin-tropomyosin regulatory complex, is required for heart muscle relaxation during the cardiac cycle. Expressed only in cardiac muscle, cTnI is widely used in the clinic as a serum biomarker of cardiac injury. In vivo function of cTnI is influenced by phosphorylation and proteolysis; therefore analysis of post-translational modifications of the intact protein should greatly facilitate the understanding of cardiac regulatory mechanisms and may improve cTnI as a disease biomarker. cTnI (24 kDa, pI~9.5) contains twelve serine, eight threonine, and three tyrosine residues presenting a challenge for unequivocal identification of phosphorylation sites and quantification of positional isomers. In this study, we employed top down electron capture dissociation (ECD) and electron transfer dissociation (ETD) mass spectrometry (MS) to unravel the molecular complexity of cTnI purified from human heart tissue. High resolution MS spectra of human cTnI revealed a high degree of heterogeneity, corresponding to phosphorylation, acetylation, oxidation, and C-terminal proteolysis. Thirty six molecular ions of cTnI were detected in a single ESI/FTMS spectrum despite running as a single sharp band on SDS-PAGE. ECD of monophosphorylated cTnI localized two major basal phosphorylation sites: a well-known site at Ser22 and a novel site at Ser76/Thr77 each with partial occupancy (Ser22: 53%; Ser76/Thr77: 36%). Top down MS3 analysis of diphosphorylated cTnI revealed occupancy of Ser23 only in diphosphorylated species consistent with sequential (or ordered) phosphorylation/dephosphorylation of the Ser22/23 pair. Top down MS of cTnI provide unique opportunities for unraveling its molecular complexity and for quantification of phosphorylated positional isomers thus allowing to establishing the relevance of such modifications to physiological functions and disease status.