The pattern of phosphorylation of adjacent serine residues in several peptides based on the N‐terminal region of human cardiac troponin I has been analysed by PAGE and 'H NMR spectroscopy to identify the products. With cAMP‐dependent protein kinase, Ser24 is rapidly phosphorylated, and subsequent much slower phosphorylation of Ser23 occurs only after phosphorylation of Ser24 is almost complete. Monophosphorylation of the peptide at Ser23 was not detected at any time. On replacement of Arg22 with Ala or Met the sole phosphorylation target was Ser23, phosphorylation being considerably slower than for Ser24 in the wild‐type peptide, while diphosphorylation could not be detected after prolonged incubation. The results emphasise the importance of the N‐terminal sequence RRRSS for the function of cardiac troponin I and imply that in human cardiac muscle unstimulated by adrenaline, troponin I is phosphorylated on Ser24. Comparative two‐dimensional NOESY data indicate that in the diphosphorylated form at physiological pH values, specific structural constraints are imposed on the N‐terminal pep‐tide region. These constraints result in the effective screening of the two phosphate groups from each other by the arginine residues N‐terminal to the serine pair and stabilisation of the structure in the region of residues 25–29, which is adjacent to a site of interaction between troponin I and troponin C. These conformational changes presumably underlie the decrease in calcium sensitivity of the myofibrillar ATPase that occurs after adrenaline intervention.