TRP proteins form nonselective cation channels, often with considerable and physiologically important permeability for Ca²⁺. Typically, the relative permeability ratios are measured in bi-ionic conditions to gain a quantitative understanding of the permeation proces. This however, does not allow to estimate how much Ca²⁺ will flow under more physiological conditions, where the free extracellular Ca²⁺ concentration is much lower than the concentration of monovalent cations and other divalent cations are present. We therefore measured the fractional Ca²⁺ current directly by loading HEK293 cells expressing TRPM3 or TRPM8 channels with large amounts of Fura-2 and recorded the current and the change in fluorescence. TRPM3 channels were stimulated with pregnenolone sulfate, while TRPM8 channels were activated by menthol. After calibration, we found that 3.5% of the current through TRPM8 channels was carried by Ca²⁺, in keeping with most other nonselective channels. The current through TRPM3 channels, however, was carried by 24% Ca²⁺, an extraordinary high value. Interestingly, this high percentage was only marginally influenced by the holding potential, but was strongly dependent on the extracellular Ca²⁺ concentration, increasing to 51% in 10 mM Ca²⁺. Removing extracellular Mg²⁺ increased the fractional Ca²⁺ current through TRPM3 to 43%. These data indicate that TRPM3 channels provide a large Ca²⁺ influx and are physiologically permeable to Mg²⁺. To investigate the molecular determinants of the large Ca²⁺ flux, we constructed chimeras between TRPM3 and TRPM1 channels by exchanging the pore region. Surprisingly, we found that the large Ca²⁺ influx was not dependent on the pore region of TRPM3.