We identified voltage-activated K+ channels in freshly dispersed smooth muscle cells from the circular layer of the canine colon in patch-clamp experiments using 200 nM charybdotoxin to suppress 270-pS Ca2+-activated K+ channels (BK channels). Three channel types were distinguished in symmetrical 140 mM KCl solutions: 19.5 +/- 1.7 pS channels (KDR1), 90.6 +/- 5.4 pS channels (KDR2) and 149 +/- 4 pS intermediate-conductance Ca2+-activated K+ channels (IK channels). All three types showed an increase in open probability with membrane depolarization. Ensemble average current from KDR1 channels inactivated with a time constant of 1.7 +/- 0.1 s at +60 mV test potential, while KDR2 and IK channels did not show inactivation. IK channels were activated by free cytoplasmic [Ca2+] (10(-6 )M) but were insensitive to 4-aminopyridine (4-AP, 10 mM) and intracellular tetraethylammonium (TEA, 1 mM). KDR1 channels were sensitive to 4-AP (10 mM) and intracellular TEA (1-10 mM) but not to Ca2+. KDR2 channels did not have a consistent pharmacological profile, suggesting that this class may be comprised of several subtypes. At +40 mV membrane potential, the catalytic subunit of protein kinase A (PKA) increased the open probability of KDR1 channels 3.4-fold and of KDR2 channels 3.9-fold, but had no effect on IK channels. In the absence of Mg-ATP, PKA did not affect channel open probabilities. At physiological membrane potentials (-60 mV) only openings of KDR1 channels could be induced by PKA, suggesting that these 4-AP-sensitive 20-pS K+ channels are primarily responsible for the cAMP-mediated hyperpolarization of colonic smooth muscle cells.