BACKGROUND AND PURPOSE: ATP-sensitive K (KATP) channels, which are composed of KIR6.x associated with sulphonylurea receptor (SUR) subunits, have been detected in native smooth muscle cells, but it is currently not known which of these is expressed in mouse vas deferens myocytes. EXPERIMENTAL APPROACH: Pharmacological and electrophysiological properties of KATP channels in mouse vas deferens myocytes were investigated using patch clamp techniques. Molecular biological analyses were performed to examine the properties of these KATP channels. KEY RESULTS: During conventional whole-cell recording, pinacidil elicited an inward current that was suppressed by glibenclamide, a sulfonylurea agent, and by U-37883A, a selective KIR6.1 blocker. When 0.3 mM ATP was added to the pipette solution, the peak amplitude of the pinacidil-induced current was much smaller than that recorded in its absence. When 3 mM UDP, GDP or ADP was included in the pipette solution, an inward current was elicited after establishment of the conventional whole-cell configuration, with potency order being UDP > GDP > ADP. These nucleoside diphosphate-induced inward currents were suppressed by glibenclamide. MCC-134, a SUR modulator, induced glibenclamide-sensitive KATP currents that were similar to those induced by 100 μM pinacidil. In the cell-attached configuration, pinacidil activated channels with a conductance similar to that of KIR6.1. Reverse transcription PCR analysis revealed the expression of KIR6.1 and SUR2B transcripts and immunohistochemical studies indicated the presence of KIR6.1 and SUR2B proteins in the myocytes. CONCLUSIONS AND IMPLICATIONS: Our results indicate that native KATP channels in mouse vas deferens myocytes are a heterocomplex of KIR6.1 channels and SUR2B subunits.