•  Neurons express many types of potassium channels that are activated by voltage but relatively little is known concerning the division of labour between different channel types in a given cell. •  Our understanding of the functional roles of Kv2 channels has been hindered by the lack of selective pharmacological agents for these channels. •  We manipulated Kv2 channel expression by transfecting pyramidal neurons with wild‐type and pore mutant channels. •  We found that reduction in functional Kv2 channels led to slower firing rates, reduced gain of firing and increased spike frequency adaptation. •  We hypothesize that Kv2 channels regulate firing by controlling membrane potential during the inter‐spike interval, which in turn regulates availability of voltage‐gated sodium channels. Abstract  The largest outward potassium current in the soma of neocortical pyramidal neurons is due to channels containing Kv2.1 α subunits. These channels have been implicated in cellular responses to seizures and ischaemia, mechanisms for intrinsic plasticity and cell death, and responsiveness to anaesthetic agents. Despite their abundance, knowledge of the function of these delayed rectifier channels has been limited by the lack of specific pharmacological agents. To test for functional roles of Kv2 channels in pyramidal cells from somatosensory or motor cortex of rats (layers 2/3 or 5), we transfected cortical neurons with DNA for a Kv2.1 pore mutant (Kv2.1W365C/Y380T: Kv2.1 DN) in an organotypic culture model to manipulate channel expression. Slices were obtained from rats at postnatal days (P7‐P14) and maintained in organotypic culture. We used biolistic methods to transfect neurons with gold ‘bullets’ coated with DNA for the Kv2.1 DN and green fluorescent protein (GFP), GFP alone, or wild type (WT) Kv2.1 plus GFP. Cells that fluoresced green, contained a bullet and responded to positive or negative pressure from the recording pipette were considered to be transfected cells. In each slice, we recorded from a transfected cell and a control non‐transfected cell from the same layer and area. Whole‐cell voltage‐clamp recordings obtained after 3–7 days in culture showed that cells transfected with the Kv2.1 DN had a significant reduction in outward current (∼45% decrease in the total current density measured 200 ms after onset of a voltage step from –78 to –2 mV). Transfection with GFP alone did not affect current amplitude and overexpression of the Kv2.1 WT resulted in greatly increased currents. Current‐clamp experiments were used to assess the functional consequences of manipulation of Kv2.1 expression. The results suggest roles for Kv2 channels in controlling membrane potential during the interspike interval (ISI), firing rate, spike frequency adaptation (SFA) and the steady‐state gain of firing. Specifically, firing rate and gain were reduced in the Kv2.1 DN cells. The most parsimonious explanation for the effects on firing is that in the absence of Kv2 channels, the membrane remains depolarized during the ISIs, preventing recovery of Na+ channels from inactivation. Depolarization and the number of inactivated Na+ channels would build with successive spikes, resulting in slower firing and enhanced spike frequency adaptation in the Kv2.1 DN cells.