In this study, we provide a direct comparison of the gating characteristics and blocker sensitivity of mouse TRPM4 and TRPM5, two Ca²⁺-activated nonselective cation channels of the melastatin related TRP (transient receptor potential) family. HEK293 cells were grown under standard tissue culture conditions and transiently transfected with a bicistronic vector containing GFP and either TRPM4 or TRPM5. Data are presented as mean ± S.E.M. Using combined whole-cell patch clamp technique and flash photolysis of caged Ca²⁺ for intracellular Ca²⁺ measurements, we investigated the dependence of current activation on the intracellular Ca²⁺ concentrations. TRPM5 currents revealed higher sensitivity to [Ca²⁺]_i than TRPM4: TRPM5 was activated by submicromolar concentrations of Ca²⁺ with values of 0.9 ± 0.13 μM (n=5) compared to the higher activation threshold of TRPM4 at values of 5.1 ± 0.7 μM (n=5). In both cases, membrane currents developed independently from the rate of changes in [Ca²⁺]_i. In cell-free inside-out patches, the concentration dependence of current activation for both channels was shifted to higher concentrations by a factor 30, but confirmed the difference in Ca²⁺ sensitivity between TRPM4 and TRPM5. Both channels revealed similar biophysical properties and are characterized by voltage-dependent activation mechanisms showing fast deactivation at negative potentials and activation at positive potentials with similar kinetics. In inside-out patches, Ca²⁺-activated TRPM4 and TRPM5 were equally sensitive to the intracellular polyamine spermine and the antimycotic clotrimazole, while TRPM4 displayed a 10-fold higher sensitivity for block by flufenamic acid with IC₅₀ of 2.8 μM (n=2-8) compared to TRPM5 (IC₅₀=24.5 μM, n=6-12). Importantly, we found a prominent difference in ATP-sensitivity between the channels: ATP^4- blocks TRPM4 with high affinity (IC₅₀=0.8 ± 0.1 μM, n=9-15), while TRPM5 is insensitive to ATP^4- at concentrations up to 1 mM. In conclusion, we provide here differences between the closely related channels TRPM4 and TRPM5, which might be helpful to functionally distinguish these channels in native cells.