Voltage-gated K+ (Kv) channels are key determinants of membrane excitability in the peripheral and in the central nervous systems, functioning to control the resting membrane potentials, the waveforms of action potentials and the repetitive firing properties of individual neurons. In addition, by influencing presynaptic neurotransmitter release and the postsynaptic responses to neurotransmitters, Kv channels regulate the integrative properties of neurons and modulate synaptic potentiation and depression. Electrophysiological studies have distinguished multiple types of Kv currents, based on differences in kinetic, voltage-dependent and/or pharmacological properties, both central and peripheral neurons. Indeed, in most neurons, multiple types of Kv channels with unique time- and voltage-dependent properties, as well as with distinct cellular and subcellular distribution patterns and functional roles, are co-expressed. Consistent with the apparent functional diversity of native neuronal Kv channels, multiple subfamilies of Kv channel pore-forming (α) subunits, as well as numerous putative Kv channel accessory subunits and regulatory proteins, have been identified and shown to be differentially expressed in neurons, as well as in other cell types. Rapidly activating, inactivating and recovering Kv currents, typically referred to as A-type Kv currents (IA), for example, have been shown to be expressed in many peripheral and central neurons and to regulate input resistances, action potential durations, repetitive firing rates and the back-propagation of action potentials into dendrites, and to modulate the responses to synaptic inputs and influence synaptic plasticity. Interestingly, Kv currents with very similar biophysical properties, referred to as Ito,f (fast transient outward Kv currents), have been identified in mammalian cardiac myocytes and shown to control the early phase of myocardial action potential repolarization. Interestingly, a number of recent studies have demonstrated critical roles for pore-forming (α) subunits of the Kv4 subfamily, particularly Kv4.2 and Kv4.3, but also Kv4.1, in the generation of native neuronal IA channels, as well as in the generation of native cardiac Ito,f channels. Studies in heterologous cells have also suggested potential functional roles for a number of transmembrane and cytosolic Kv channel accessory proteins, including members of the minK/MiRP (minimal K+ channel), KChIP (K+ channel interacting protein) and DPP (dipeptidyl peptidase) subfamilies of Kv channel accessory subunits, in the generation of Kv4-encoded channels. Although the results of recent studies using targeted “knockdown” and “knockout” strategies to define directly the physiological role(s) Kv4 channel accessory subunits in determining the functional cell surface expression and the biophysical properties of native Kv4-encoded neuronal IA (and myocardial Ito,f) channels are consistent with some of the results obtained in heterologous overexpression studies, novel physiological roles have also been revealed. In addition, the application of quantitative mass spectrometry-based proteomic analysis as a rapid and unbiased approach to identify the components of native Kv4-encoded neuronal IA (and myocardial Ito,f) channel macromolecular protein complexes has, in addition to confirming association between Kv4 α subunits and the KChIPx and DPPx accessory subunits in brain, also identified a number of novel Kv channel accessory/regulatory proteins. Multiple experimental approaches, exploited in parallel to define the physiological roles of newly identified, putative channel accessory and regulatory subunits in the generation of native neuronal IA channels, will also be discussed.