The present study describes the single channel properties of a novel cGMP-activated Ca2+-dependent Cl- channel in rat mesenteric artery smooth muscle cells. Single channel currents were recorded in cell-attached patches in the presence of 8Br cGMP in response to addition of caffeine or NA and in both outside-out and inside-out patches when the internal patch surface was bathed in cGMP and Ca2+. The channels were permeable to Cl- ions with an anion permeability sequence of SCN- (1.7) > Cl- (1.0) > I- (0.6). Single channel NPo was independent of voltage and the channels displayed three conductance levels of 15 pS, 35 pS and 55 pS. cGMP was required for channel activation and the single channel NPo increased sharply with raised [Ca2+]i with maximal activation occurring at a [Ca2+]i of about 100 nM. The relationship between NPo and cGMP concentration was voltage-independent and could be fitted by the Hill equation giving a Kd of about 3 µM and a Hill coefficient (nH) of 3. cGMP- and Ca2+-dependent channel currents were inhibited by 10 µM ZnCl2 but niflumic acid, an inhibitor of Ca2+-activated Cl- channels, had no effect. Inhibition of cGMP-dependent protein kinase activity by the cGMP-dependent protein kinase inhibitor KT5823 or replacement of ATP by AMP-PNP reduced NPo while activation of cGMP-dependent protein kinase by 8Br PET cGMP produced a significant increase in single channel NPo. It is likely that these single channel currents underlie the noradrenaline-activated inward current important for vasomotion in these resistance arteries.