The tescalcin gene is preferentially expressed during mouse testis differentiation. Here, we demonstrate that this gene encodes a 24 kDa Ca²⁺- and Mg²⁺-binding protein with one consensus EF-hand and three additional domains with EF-hand homology. Equilibrium dialysis with ⁴⁵Ca²⁺ revealed that recombinant tescalcin binds approximately one Ca²⁺ ion at physiological concentrations (pCa 4.5). The intrinsic tryptophan fluorescence of tescalcin was significantly reduced by Ca²⁺, indicative of a conformational change. The apparent K_d for Ca²⁺ was 0.8 μM. A point mutation in the consensus EF-hand (D123A) abolished ⁴⁵Ca²⁺ binding and prevented the fluorescence quenching, demonstrating that the consensus EF-hand alone mediates the Ca²⁺-induced conformational change. Tescalcin also binds Mg²⁺ (K_d 73 μM), resulting in a much smaller fluorescence decrease. In the presence of 1 mM Mg²⁺, tescalcin's Ca²⁺ affinity is shifted to 3.5 μM. These results illustrate that tescalcin should bind Mg²⁺ constitutively in a quiescent cell, replacing it with Ca²⁺ during stimulation. We also show that tescalcin is most abundant in adult mouse heart, brain, and stomach, as well as in HeLa and HL-60 cells. Immunofluorescence microscopy revealed that tescalcin is present in the cytoplasm and nucleus, with concentration in membrane ruffles and lamellipodia in the presence of serum, where it colocalizes with the small guanosine triphosphatase Rac-1. Tescalcin shares sequence and functional homology with calcineurin-B homologous protein (CHP), and we found that tescalcin, like CHP, can inhibit the phosphatase activity of calcineurin A. Hence, tescalcin is a novel calcineurin B-like protein that binds a single Ca²⁺ ion.