Transient outward rectifying conductances or A-like conductances in sympathetic preganglionic neurons (SPN) are prolonged, lasting for hundreds of milliseconds to seconds and are thought to play a key role in the regulation of SPN firing frequency. Here, a multidisciplinary electrophysiological, pharmacological and molecular single-cell rt-PCR approach was used to investigate the kinetics, pharmacological profile and putative K+ channel subunits underlying the transient outward rectifying conductance expressed in SPN. SPN expressed a 4-aminopyridine (4-AP) sensitive transient outward rectification with significantly longer decay kinetics than reported for many other central neurons. The conductance and corresponding current in voltage-clamp conditions was also sensitive to the Kv4.2 and Kv4.3 blocker phrixotoxin-2 (1-10 μM) and the blocker of rapidly inactivating Kv channels, pandinotoxin-Kα (50 nM). The conductance and corresponding current was only weakly sensitive to the Kv1 channel blocker tityustoxin-Kα and insensitive to dendrotoxin I (200 nM) and the Kv3.4 channel blocker BDS-II (1 μM). Single-cell RT-PCR revealed mRNA expression for the α-subunits Kv4.1 and Kv4.3 in the majority and Kv1.5 in less than half of SPN. mRNA for accessory β-subunits was detected for Kvβ2 in all SPN with differential expression of mRNA for KChIP1, Kvβ1 and Kvβ3 and the peptidase homologue DPP6. These data together suggest that the transient outwardly rectifying conductance in SPN is mediated by members of the Kv4 subfamily (Kv4.1 and Kv4.3) in association with the β-subunit Kvβ2. Differential expression of the accessory β subunits, which may act to modulate channel density and kinetics in SPN, may underlie the prolonged and variable time-course of this conductance in these neurons.