Potassium channels are extremely diverse regulators of neuronal excitability. As part of an investigation into how this molecular diversity is utilized by neurones, we examined the expression and biophysical properties of native Kv1 channels in layer II/III pyramidal neurones from somatosensory and motor cortex. Single-cell RT-PCR, immunocytochemistry, and whole cell recordings with specific peptide toxins revealed that individual pyramidal cells express multiple Kv1 α-subunits. The most abundant subunit mRNAs were Kv1.1 > 1.2 > 1.4 > 1.3. All of these subunits were localized to somatodendritic as well as axonal cell compartments. These data suggest variability in the subunit complexion of Kv1 channels in these cells. The α-dendrotoxin (α-DTX)-sensitive current activated more rapidly and at more negative potentials than the α-DTX-insensitive current, was first observed at voltages near action potential threshold, and was relatively insensitive to holding potential. The α-DTX-sensitive current comprised about 10% of outward current at steady-state, in response to steps from −70 mV. From −50 mV, this percentage increased to ∼20%. All cells expressed an α-DTX-sensitive current with slow inactivation kinetics. In some cells a transient component was also present. Deactivation kinetics were voltage dependent, such that deactivation was slow at potentials traversed by interspike intervals during repetitive firing. Because of its kinetics and voltage dependence, the α-DTX-sensitive current should be most important at physiological resting potentials and in response to brief stimuli. Kv1 channels should also be important at voltages near threshold and corresponding to interspike intervals.