ABA stimulation of outward K+ current (IK,out) in Vicia faba guard cells has been correlated with a rise in cytosolic pH (pHi). However, the underlying mechanism by which IK,out is affected by pHi has remained unknown. Here, we demonstrate that pHi regulates outward K+ current in isolated membrane patches from Vicia faba guard cells. The stimulatory effect of alkalinizing pHi was voltage insensitive and independent of the two free calcium levels tested, 50 nM and 1 microM. The single-channel conductance was only slightly affected by pHi. Based on single-channel measurements, the kinetics of time-activated whole-cell current, and the analysis of current noise in whole-cell recordings, we conclude that alkaline pHi enhances the magnitude of IK,out by increasing the number of channels available for activation. The fact that the pHi effect is seen in excised patches indicates that signal transduction pathways involved in the regulation of IK,out by pHi, and by implication, components of hormonal signal transduction pathways that are downstream of pHi, are membrane-delimited.