We investigated the properties of K(+) channels in the basolateral membrane of the cortical thick ascending limb (CTAL) using the patch-clamp technique. Approximately 34% of cell-attached patches contained an inwardly rectifying K(+) channel (K(+)-to-Na(+) permeability ratio approximately 22), having an inward conductance (G(in)) of 44 pS and an outward conductance (G(out)) of approximately 10 pS (G(in)/G(out) approximately 4). Channel activity (NP(o)) increased with depolarization. When the cytosolic sides of inside-out patches were exposed to an Mg(2+)-free medium, the channel had a G(in) of 50 pS and was weakly inwardly rectifying (G(in)/G(out) approximately 1). Cytosolic Mg(2+) reduced G(out), yielding a G(in)/G(out) of 3.8 at 1.3 mM Mg(2+). Internal Na(+) also yielded a G(in)/G(out) of 1.6 at 20 mM Na(+). Spermine reduced NP(o) on inside-out membrane patches. Sensitivity to spermine at depolarizing voltages [half-maximal inhibitory concentration (K(i)) = 0.2 microM] was much greater than at hyperpolarizing voltages (K(i) = 26 microM). Half-inactivation by 0.5 microM spermine occurred at a clamp potential of 43 mV, with an effective valence of 1.25. A sigmoid relationship between bath pH and NP(o) of inside-out membrane patches was observed, with a pK of 7.6 and a Hill coefficient of 1.8. Intracellular acidification also reduced the NP(o) of cell-attached patches. This channel is probably a major component of K(+) conductance in the CTAL basolateral membrane.