We present a comprehensive profile of amino acid side-chain constraints in a calmodulin (CaM) peptide complex. These data were obtained from the analysis of calmodulin binding to an array of all single substitution analogues as well as N- and C-terminal truncations of the skMLCK derived M13 peptide ligand. The experimentally derived binding data were evaluated with respect to the known 3D-structure of the CaM/M13 complex. Besides an almost perfect agreement between the measured affinities and the structural data, the unexpected high-affine Asn5Ala variant of the M13(*) peptide described by Montigiani et al. could be verified. In contrast to other reports our data clearly support the postulate of the minor and major hydrophobic anchors of this calcium dependent interaction.