The expression and functional role of ether-a-go-go-related gene (erg) K⁺ channels were examined in the bovine epididymal duct. Sperm transit through the epididymal duct relies on spontaneous phasic contractions (SC) of the peritubular smooth muscle wall. Isometric tension studies revealed SC-enhancing effects of the erg channel blockers E-4031, dofetilide, cisapride and haloperidol and SC-suppressing effects of the HERG activator NS1643. In the corpus epididymidis, EC₅₀ values of 32 nM and 8.3 µM were determined for E-4031 and NS1643, respectively. E-4031 was also able to elicit contraction in epithelium-denuded corpus segments, which lacked SC. In the cauda region, E-4031 and NS1643 exerted effects on agonist-induced contraction similar to those observed in the proximal duct. Experiments with nifedipine and thapsigargin suggested that the excitatory effects of E-4031 depended mainly on external calcium influx and not on intracellular calcium release. Western blot and RT-PCR assays revealed the expression of both, erg1a and erg1b, in all duct regions. Since erg1b appears to predominate in the epididymal duct, patch-clamp experiments were performed on heterologously expressed erg1b channels to investigate the sensitivity of this splice variant to NS1643. In contrast to its effects on erg1a, NS1643 induced a concentration-dependent current increase mainly due to a marked leftward-shift in erg1b channel activation by about 30 mV at 10 µM, explaining the inhibitory effect of the drug on epididymal SC. In summary, these data provide strong evidence for a physiological role of erg1 channels in regulating epididymal motility patterns.