Recently a novel cGMP-activated Ca²⁺-dependent Cl^- channel has been described in rat mesenteric artery smooth muscle cells. In the present work we have investigated the actions of calmodulin (CaM) on single I_Cl(cGMP,Ca) channel currents in inside-out patches. When 1 µM CaM was applied to the intracellular surface of inside-out patches bathed with 10 µM cGMP and 100 nM [Ca²⁺]_i there was approximately a ten-fold increase in channel activity (NP_o). This effect of CaM was not observed with lower [Ca²⁺]_i and 100 nM [Ca²⁺]_i with 1 µM CaM did not activate Cl^- channels in the absence of cGMP. The unitary conductance, reversal potential and mean open time of the single-channel currents were similar in the absence or presence of CaM. With 10 µM cGMP and 100 nM [Ca²⁺]_i the relationship between NP_o and CaM concentration was well fitted by the Hill equation yielding an equilibrium constant for CaM of about 1.9 nM and a Hill coefficient of 1.7. With 1 µM CaM (+ 10 µM cGMP) the relationship between [Ca²⁺]_i and NP_o was also fitted by the Hill equation which yielded an apparent equilibrium constant of 74 nM [Ca²⁺]_i and a Hill coefficient of 4.8. When [Ca²⁺]_i was increased from 300 nM to 1 µM there was a decrease in NP_o. The potentiating effect of CaM was markedly reduced by the selective CaM binding peptide Trp (5 nM) but not by the Ca²⁺/CaM-dependent protein kinase II (CaMKII) inhibitor autocamtide II related inhibitory peptide (AIP). It is concluded that CaM potentiates the activity of single I_Cl(cGMP,Ca) channel currents by increasing the probability of channel opening via a CaMKII-independent mechanism.