1. The Na(+)-activated K+ channel current was recorded from inside-out membrane patches excised from single ventricular cells of guinea-pig hearts. 2. The single channel current-voltage relations showed inward-going rectification with an asymptotic conductance of 180-210 pS for the inward current at 150 mM [K+]o, when [K+]i was changed between 5.4 and 150 mM. The reversal potential indicated the PNa/PK of about 0.02. 3. The amplitude of outward current was reduced by increasing [Mg2+]i or [Na+]i, but no obvious blocking noise was recorded. The outward current, which remained shortly after quick removal of both [Na+]i and [Mg2+]i, revealed an ohmic conductance of the K+ channel. 4. The [Mg2+]i and [Na+]i block was increased e-fold by depolarizing the membrane by 49 mV, while the inward current was not blocked. 5. The Na(+)-activated K+ channel showed frequent subconductance levels. The variance-mean analysis resolved at least ten major sublevels. The density distribution of the sublevels were measured by composing the conventional amplitude histogram, excluding clear closed state currents, and then dividing the histogram into five segments. The probability of staying in each segment (Pn) was almost always voltage independent, and the grand averages were P1 = 9.5 +/- 5.9%, P2 = 6.3 +/- 2.1%, P3 = 4.2 +/- 1.8%, P4 = 7.8 +/- 2.5%, and P5 = 39.3 +/- 5.6%, from the lowest segment, respectively. 6. The values of Pn in partially blocked conditions by Na+ and Mg2+ (outward current) were not clearly different from those without any channel block (inward current). The values of Pn, measured before and after applying Ba2+ in the pipette, were also very similar. 7. The above findings indicate that the inward-going rectification of the Na(+)-activated K+ channel is due to the Na+ and Mg2+ block. The subconductance of the channel is not due to any channel block by Na+ or Mg2+, but may be attributable to multiple open states of a single-barrel channel, which has a large conductance. The channel may be blocked from any open conformation with an equal probability and with very fast kinetics.