The inward K+ channels (IKin) of guard cells are inhibited upon application of abscisic acid (ABA). It has been postulated that I(Kin) inhibition requires an elevation in cytosolic free Ca2+ levels ([Ca2+]c) because: (i) experimental increases in [Ca2+]c can mimic the ABA effect, and; (ii) ABA can trigger an elevation of [Ca2+]c in guard cells. However, not all guard cells respond to ABA with a [Ca2+]c increase, and the magnitude of the increases that do occur is variable. Therefore, an obligate role for Ca2+ in the regulation of downstream effectors of ABA response, such as the I(Kin) channels, remains in question. In this study, we developed a methodology for simultaneous patch clamping and confocal ratiometric Ca2+ imaging of Vicia faba L. guard-cell protoplasts. This allowed us to directly assess the relationship between ABA-induced changes in [Ca2+]c and I(Kin) inhibition. In the presence of extracellular Ca2+, the extent of [Ca2+]c elevation correlated with the extent of I(Kin) inhibition. However, upon chelation of either extracellular Ca2+, [Ca2+]c or both, extracellular Ca2+ and [Ca2+]c, [Ca2+]c elevation did not occur in response to ABA yet I(Kin) currents were still strongly inhibited. These data illustrate that Ca2+-independent regulation is involved in ABA-inhibition of stomatal opening processes.