S100 proteins became of major interest because of their divergent cell- and tissue-specific expression, their close association with a number of human diseases, and their importance for clinical diagnostics. Here, we report for the first time the purification and characterization of human recombinant S100A13. Flow dialysis revealed that the homodimeric S100A13 binds four Ca(2+) in two sets of binding sites, both displaying positive cooperativity but of very different affinity. Fluorescence and difference spectrophotometry indicate that the Trp/Tyr signal changes are almost complete upon binding of Ca(2+) to the two high affinity sites, which probably correspond to the C-terminal EF-hands in each subunit. The far-UV circular dichroic signal also changes upon binding of the first two Ca(2+). So far, the tissue distribution of S100A13 has not been well characterized. Here, we show that S100A13 is widely expressed in various types of tissues with a high expression level in thyroid gland. Using specific antisera against S100A13, high protein expression was detected in follicle cells of thyroid, Leydig cells of testis, and specific cells of brain. In human smooth muscle cells, which co-express S100A2 in the nucleus and S100A1 in stress fibers, S100A13 shows a unique subcellular localization in the perinuclear area. These data suggest diverse functions for this protein in signal transduction.