Rabbit urethral smooth muscle cells (SMC) express a delayed rectifer K+ current, but its molecular identity is unknown. The purpose of the present study was to identify which Kv channel subtype encodes this channel in these cells. Rabbits were humanely killed with pentobarbitone (I.V.) and the urethra was removed. For electrophysiology experiments, SMC were isolated as previously described (Sergeant et al. 2000), perfused with Hanks’ solution at 37 °C and studied under voltage clamp using the perforated patch configuration with K+-rich pipette solutions. Following successful perforation of the membrane, cells were bathed in Mg-substituted Ca2+ free Hanks solution with 5 mM EGTA containing 100 nM penitrem A (to block Ca2+ and Ca2+ activated currents). Freshly dispersed urethral SMC were held at -80 mV and depolarized to +40 mV for 500 ms to evoke the Kv currents. These were inhibited by TEA in a concentration dependent manner (IC50 1.8 ± 0.7 mM, n=8), but were unaffected by either margatoxin or α-dendrotoxin (100 nM, n=3), suggesting that they were not encoded by Kv1.1, Kv1.2, Kv1.3 or Kv1.6 channels. However, in 7 experiments application of the Kv2 selective blocker, stromatoxin inhibited the currents in a concentration dependent manner (IC50=201 ± 39 nM) consistent with the idea that they were encoded by Kv2 channels. We next cloned Kv2.1 and Kv2.2, channels from the rabbit urethra and stably expressed them in human embryonic kidney (HEK) cells. These cells were studied using the whole cell configuration of the patch clamp technique with the same protocol as above. Kv2.1 and Kv2.2 currents were blocked in a concentration dependent manner by TEA with IC50s of 3.9 ± 0.75 mM and 10 ± 3.5 mM (n=6). Stromatoxin also inhibited these currents with IC50 of 66 ± 18 nM (Kv2.1, n=5) and 49 ± 19.5 nM (Kv2.2, n=6). A two-pulse protocol was used to assess the voltage dependent inactivation in cells expressing either Kv2.1 or Kv2.2 channels. The protocol consisted of holding the cells at -60 mV and stepping to conditioning potentials ranging from -100 to +40 mV for 10 s before stepping to a +40 mV test pulse for 500ms. In 13 cells transfected with Kv2.1 only, the mean V1/2 of inactivation was -55 ± 3 mV compared to -30 ± 3 mV in the cells transfected with Kv2.2 (n=11). When native SMC were studied with the same protocol, the V1/2 of inactivation of the Kv current was -56 ± 3 mV (n=9). These electrophysiological and pharmacological data suggest that Kv2.1 channels underlie the delayed rectifier Kv current in rabbit urethral SMC.