Large-conductance, Ca(2+)-activated K+ channels were identified in single smooth muscle cells freshly isolated from rabbit superior mesenteric artery. They typically showed a reversal potential close to 0 mV in excised, inside-out patches in symmetric 130 mmol/L [K+] with a unitary conductance of 260 pS, and increased activity at more positive potentials and/or when [Ca2+] was raised at the cytosolic surface of the membrane. Both in cell-attached and in excised, inside-out configurations, stretching the membrane patch by applying suction to the back of the patch pipette increased the activity of these channels without changing either the unitary conductance or the voltage sensitivity of the channel. Stretch activation was repeatedly seen in inside-out patches when both surfaces were bathed with a 0 Ca2+ solution containing 2 or 5 mmol/L EGTA to chelate trace amounts of Ca2+, making it highly improbable that stretch activation could be secondary to a stretch-induced flux of Ca2+. Consequently, stretch activation of large-conductance, Ca(2+)-activated K+ channels in mesenteric artery smooth muscle cells seems to be due to a direct effect of stretch on the channel itself or on some closely associated, membrane-bound entity.