1. ROMK1 potassium channel currents were examined in Xenopus oocytes by two-microelectrode voltage-clamp and patch-clamp techniques following injection of oocytes with in vitro transcribed ROMK1 cRNA. Macroscopic currents recorded from intact cells rectified inwardly at positive potentials. 2. In inside-out membrane patches rectification is manifested as an apparent reduction of single channel current (at 500 Hz) in the presence of 0.1-10 mM Mg2+, without a decrease in the channel open probability. No inward rectification is observed when membrane patches are isolated into solutions containing potassium as the only internal inorganic cation. 3. Mg2+ block can be described by a simple one-site model for Mg2+ binding with K0 ([Mg2+] causing half-maximal block at 0 mV) of 16.7 mM and delta (the fraction of the membrane field sensed by the blocking Mg2+) of 0.35. 4. The voltage dependence of channel block by cytoplasmic Mg2+ was shifted approximately -50 mV by a reduction in extracellular [K+] from 140 to 0 mM, corresponding to a decrease of K0 to 4.4 mM. 5. At negative membrane potentials, ROMK1 channels exhibit a single subconducting state that is approximately 4/10 of the full conductance. The incidence of subconductance states is not appreciably enhanced in the presence of Mg2+.