A novel member of the S100 protein family, present in human placenta, has been characterized by protein sequencing, cDNA cloning, and analysis of Ca2+-binding properties. Since the placenta protein of 95 amino acid residues shares about 50% sequence identify with the brain S100 proteins α and β, we proposed the name S100P. The cDNA was expressed in Escherichia coli and recombinant S100P was purified in high yield. S100P is a homodimer and has two functional EF hands/plypeptide chain. The low-affinity site (Kd= 800 μM), which, in analogy to S100β, seems to involve the N terminal EF hand, can be followed by the Ca2+ -dependent decrease in tyrosine fluorescence. The high-affinity site., provided by the C-terminal EF hand, influences the reactivity of the sole cysteine which is located in the C-terminal extension (Cys85). Binding to the high-affinity site (Kd= 1.6 μM) can be monitored by fluorescence spectroscopy of S100P labelled at Cys85 with 6-proprionly-2-dimethylaminonaphtalene (Prodan). The Prodan fluorescence shows a Ca2+ -dependent red shift of the maximum emisson wavelength from 485 nm to 502 nm, which is accomplished by an approximately twofold loss in integrated fluorescence intensity. This indicates that Cys85 becomes more exposed to the solvant in Ca2+ -bound S100P, making this region of the molecule, the so-called C-terminal extention with a cellular target. In p11, a different member of the S100 family, C-terminal extension which contains a corresponding cysteine (Cys82 in p11), is involved in a Ca2+ -independent complex formation with the protein ligand annexin II. The combined results support the hypothesis that S100 proteins interact in general with their targets after a Ca2+ -dependent conformational change which involves hydrophobic residues of the C-terminal extension.