Biophysical properties of ROMK2 channel were investigated at physiological temperature, after reexpression of the recombinant ROMK2 protein in a mammalian cell expression system (COS-7). We observed that ROMK2 induced an inwardly rectifying K+ current whether polyvalent cations were present or not. Above +10 mV, ROMK2-induced current exhibited a voltage- and time-dependent decay, consistent with an inactivation process. Inactivation of ROMK2-induced current was also seen in inside out patch from ROMK2-expressing Xenopus oocyte. In COS-7 cells, inactivation was found to account for most of the inward rectification. Mg2+ and spermine modulated rectification by accelerating inactivation kinetics independently of membrane potential. These results establish for the first time ROMK2 properties in a mammalian cell expression system.