The inwardly rectifying properties and molecular basis of ATP-sensitive K⁺ channels (K_ATP channels) have now been established for several cell types, however, these aspects of non-vascular smooth muscle K_ATP channels still remain to be defined. In this study, we investigated the molecular basis of the pore of K_ATP channels of pig urethral smooth muscle cells through a comparative study of the inwardly rectifying properties, conductance and regulation by protein kinase C (PKC) of native and homo- and hetero-concatemeric recombinant Kir6.x channels co-expressed with SUR2B in human embryonic kidney (HEK) 293 cells by patch-clamp technique (conventional whole-cell and cell-attached modes). In conventional whole-cell clamp recordings, levcromakalim ( 1 µM) caused a concentration-dependent increase in current that demonstrated strong inward rectification at positive membrane potentials. In cell-attached mode, the unitary amplitude of levcromakalim-induced native and recombinant hetero-concatemeric Kir6.1-Kir6.2 K_ATP channels also showed strong inward rectification at positive membrane potentials. Phorbol 12,13-dibutyrate (PdBu), but not the inactive phorbol ester, 4-phorbol 12,13-didecanoate (4-PDD), enhanced the activity of native and hetero-concatemeric K_ATP channels at -50 mV. The conductance of the native channels at ~43 pS was consistent with that of hetero-concatemeric channels with a pore-forming subunit composition of (Kir6.1)_3-(Kir6.2). RT-PCR analysis revealed the expression of Kir6.1 and Kir6.2 transcripts in pig urethral myocytes. Our findings provide the first evidence that the predominant K_ATP channel expressed in pig urethral smooth muscle possess a unique, heteromeric pore structure that differs from the homomeric Kir6.1 channels of vascular myocytes and is responsible for the differences in inward rectification, conductance and PKC regulation exhibited by the channels in these smooth muscle cell types.