Human calmodulin-like protein (CLP) is closely related to vertebrate calmodulin, yet its unique cell specific expression pattern, overlapping but divergent biochemical properties, and specific target proteins suggest that it is not an isoform of calmodulin. To gain insight into the structural differences that may underlie the difference target specificities and biochemical properties of CLP when compared to calmodulin, we determined the sequential backbone assignment and associated secondary structure of 144 out of the 148 residues of Ca2+-CLP by using multinuclear multidimensional NMR spectroscopy. Despite a very high overall degree of structural similarity between CLP and calmodulin, a number of significant differences were found mainly in the length of alpha-helices and in the central nonhelical flexible region. Interestingly, the regions of greatest primary sequence divergence between CLP and calmodulin in helices III and VIII displayed only minor secondary structure differences. The data suggest that the distinct differences in target specificity and biochemical properties of CLP and calmodulin result from the sum of several minor structural and side-chain changes spread over multiple domains in these proteins.