Previous studies have revealed the presence of four kinetically distinct voltage-gated K+ currents, I(Af), I(As), I(K), and I(SS), in rat superior cervical ganglion (SCG) neurons and demonstrated that I(K) and I(SS) are expressed in all cells, whereas I(Af) and I(As) are differentially distributed. Previous studies have also revealed the presence of distinct components of I(Af) encoded by alpha-subunits of the Kv1 and Kv4 subfamilies. In the experiments described here, pore mutants of Kv2.1 (Kv2.1W365C/Y380T) and Kv2.2 (Kv2.2W373C/Y388T) that function as Kv2 subfamily-specific dominant negatives (Kv2.1DN and Kv2.2DN) were generated to probe the functional role(s) of Kv2 alpha-subunits. Expression of Kv2.1DN or Kv2.2DN in human embryonic kidney-293 cells selectively attenuates Kv2.1- or Kv2.2-encoded K+ currents, respectively. Using the Biolistics Gene Gun, cDNA constructs encoding either Kv2.1DN or Kv2.2DN [and enhanced green fluorescent protein (EGFP)] were introduced into SCG neurons. Whole-cell recordings from EGFP-positive Kv2.1DN or Kv2.2DN-expressing cells revealed selective decreases in I(K). Coexpression of Kv2.1DN and Kv2.2DN eliminates I(K) in most (75%) SCG cells and, in the remaining (25%) cells, I(K) density is reduced. Together with biochemical data revealing that Kv2.1 and Kv2.2 alpha-subunits do not associate in rat SCGs, these results suggest that Kv2.1 and Kv2.2 form distinct populations of I(K) channels, and that Kv2 alpha-subunits underlie (most of) I(K) in SCG neurons. Similar to wild-type cells, phasic, adapting, and tonic firing patterns are evident in SCG cells expressing Kv2.1DN or Kv2.2DN, although action potential durations in tonic cells are prolonged. Expression of Kv2.2DN also results in membrane depolarization, suggesting that Kv2.1- and Kv2.2-encoded I(K) channels play distinct roles in regulating the excitability of SCG neurons.