Reconstitution studies of a protein from domain fragments can furnish important insights into the distinctive role of particular domain interactions and how they affect biophysical properties important for function. Using isothermal titration calorimetry (ITC) and a number of spectroscopic and chromatographic tools, including CD, fluorescence and NMR spectroscopy, size-exclusion chromatography and non-denaturing agarose gel electrophoresis, we have investigated the reconstitution of the ubiquitous Ca2+-sensor protein calmodulin (CaM) and its globular domains from fragments comprising one or two EF-hands. The studies were carried out with and without the target peptide from smooth muscle myosin light chain kinase (smMLCKp). The CaM-target complex can be reconstituted from the three components consisting of the target peptide and the globular domains TR1C and TR2C. In the absence of peptide, there is no evidence for association of the globular domains. The globular domains can further be reconstituted from their corresponding native subdomains. The dissociation constant, K(D), in 2 mM Tris-HCl (pH 7.5), for the subdomain complexes, EF1:EF2 and EF3:EF4, was determined with ITC to 9.3 x 10(-7) M and 5.9 x 10(-8) M, respectively. Thus, the affinity between the two C-terminal subdomains, located within TR2C, is stronger by a factor of 16 than that between the corresponding subdomains within TR1C. These observations are corroborated by the spectroscopic and chromatographic investigations.