TRPM4b (in contrast to the short splice variant TRPM4a) is a Ca²⁺-activated but Ca²⁺-impermeable cation channel. We have studied TRPM4 currents in inside-out patches. Supramicromolar Ca²⁺ concentrations applied at the inner side, [Ca²⁺]_i, activated TRPM4 with an EC₅₀ value of 0.37 mM, a value that is much higher than that of whole-cell currents. Current amplitudes decreased above 1 mM [Ca²⁺]_i, (IC₅₀ 9.3 mM). Sr²⁺ but not Ba²⁺could partially substitute for Ca²⁺. ATP, ADP, AMP and AMP-PNP all quickly and reversibly inhibited TRPM4 with IC₅₀ values between 2 and 19 μM (at +100 mV). Adenosine also blocked TRPM4 at 630 μM. The block at high ATP concentrations was incomplete and was not affected by the presence of free Mg²⁺. ADP induced the most sensitive block with an IC₅₀ of 2.2 μM. For inhibition of TRPM4 by free ATP⁴⁻, an IC₅₀ value of 1.7±0.3 μM was calculated. GTP, UTP and CTP at concentrations up to 1 mM did not induce a similar block. Spermine blocked TRPM4 currents with an IC₅₀ of 61 μM. In conclusion, TRPM4 is a channel that can be effectively modulated by intracellular nucleotides and polyamines.