Cytosolic calcium-binding proteins termed calbindins are widely regarded as a key component of the machinery used to transport calcium safely across cells. Acting as mobile buffers, calbindins are thought to ferry calcium in bulk and simultaneously protect against its potentially cytotoxic effects. Here, we contradict this dogma by showing that teeth and bones were produced normally in null mutant mice lacking calbindin 28kDa . Structural analysis of dental enamel, the development of which depends critically on active calcium transport, showed that mineralization was unaffected in calbindin 28kDa -null mutants. An unchanged rate of calcium transport was verified by measurements of 45 Ca incorporation into developing teeth in vivo. In enamelforming cells, the absence of calbindin 28kDa was not compensated by other cytosolic calcium-binding proteins as detectable by 45 Ca overlay, two-dimensional gel, and equilibrium binding analyses. Despite a 33% decrease in cytosolic buffer capacity, cytotoxicity was not evident in either the null mutant enamel or its formative cells. This is the first definitive evidence that calbindins are not required for active calcium transport, either as ferries or as facilitative buffers. Moreover, in challenging the broader notion of a cytosolic route for calcium, the findings support an alternative paradigm involving passage via calcium-tolerant organelles.The active transport of calcium across cells holds widespread importance in medicine and biology, yet the underlying mechanisms remain unclear. Operating in many places (e.g. gut, kidney, placenta, teeth, bones, oviduct, lung, inner ear), active transport is used to control the amount of calcium in body fluids and so impacts on nutrition, biomineralization, fertility, respiration, and hearing (1, 2). Superior control is achieved by passaging calcium actively through cells rather than passively between them, but this comes at the risk of cytotoxicity should the ability to regulate intracellular calcium be overburdened.Mechanistically, active transport is considered in three steps: the entry of calcium to the cell, transit across it, and extrusion at the other side. The transit step has received the most attention over several decades, being considered rate-limiting and having key molecular players defined. However, recent molecular characterization of calcium entry channels has transformed the field by providing a new mechanistic focus for vitamin D-restricted transport (3, 4). With these advances reigniting interest in therapeutic applications, it is important to revisit what happens following calcium entry.The 30-year-old paradigm that calcium is ferried through cytosol by mobile calcium-binding proteins (calbindins) remains widely accepted (3-9). Calbindins are thought to facilitate the naturally poor diffusion of calcium in cytosol and simultaneously buffer calcium at safe concentrations. Comprehensively supporting this view, tight correlations between calbindin expression and vitamin D-dependent transport were found in intestine...