It is well established that troponin I is a phosphoprotein. Phosphorylation alters its functional properties and this modulation of function through the action of kinases and phosphatases plays a role in tuning the contractile apparatus. The prime example of this is phosphorylation by protein kinase A (PKA) as part of inotropic and lusitropic responses to β-adrenergic stimulation. What is considerably less certain is the ‘where’ and ‘when’ of these phosphorylation processes in the human heart. Recent research has addressed this question with new techniques and the results have been surprising and somewhat disconcerting. Quantitative measurements of total phosphorylation by phosphate affinity sodium dodecylsulphate–polyacrylamide gel electrophoresis (SDS–PAGE) indicate that 1.6 mol of Pi are incorporated per mole of troponin I in the donor heart. According to current literature, troponin I is phosphorylated in vitro by PKA at Ser22 and 23, by protein kinase C (PKC) at Ser41, Ser43 and Thr142 and by PAK1 and AMPK at Ser149. Both phosphate affinity SDS–PAGE and Fourier transform mass spectrometry plus ECD show that troponin I is mostly bis-phosphorylated and that three-quarters of the bis-phosphorylated species is phosphorylated at Ser22 and 23, the PKA-specific sites. Somewhat surprisingly, there is no evidence of phosphorylation at Ser41, Ser43, Thr142 or Ser149 in human or rat heart muscle and the remaining phosphorylation is at Ser76 or Thr77 (the mass spectrometry techniques do not yet distinguish between the two). In end-stage failing heart muscle the level of phosphorylation is reduced to one-sixth of the donor level, therefore hypotheses that invoke PKC phosphorylation of troponin I need to be revised.