Small conductance Ca2+-activated K+ (SK) channels are expressed throughout the soma and dendrites of hippocampal pyramidal neurons, where they participate in the local regulation of membrane excitability and synaptic signals. Using selective SK channel inhibitors and enhancers, we recently described an inter-play between SK and L-type Ca2+ channels in the regulation of Ca2+ influx triggered in the proximal dendrites by back-propagating action potentials (bAP) (1). Besides affecting the amplitude of Ca2+ transients, SK channel inhibition also prolonged their duration, but the role of Ca2+ clearance mechanisms and their link to SK channel activity was not established. In this work, we used the Na+/Ca2+ exchanger (NCX) inhibitor benzamil to examine the interaction between Ca2+ extrusion and SK channels in the regulation of dendritic Ca2+ signals. Trains of four suprathreshold depolarizing steps (20 Hz) were delivered in the whole-cell patch clamp configuration to the somata of Fluo-4 loaded rat pyramidal neurons in dissociated cultures, prepared in accordance with UK Home Office regulations. This stimulus triggered bAPs and voltage-driven Ca2+ transients monitored by 2-photon Ca2+ imaging in the initial 50 μm of the apical dendrite. Consistent with an NCX-dependent Ca2+ clearance from the dendrites in these neurons, benzamil application prolonged the duration of the Fluo-4 signals (160.1 ± 18.8% of control; n=6, p=0.044). However, a significant increase in the amplitude of Ca2+ transients (117.6 ± 6.4% of control; n=6, p=0.017) was also detected, similar to what we had observed with SK channel blockers (1), suggesting that benzamil might act as a SK channel inhibitor. This hypothesis was tested on both neuronal SK currents and heterologously expressed SK channels. The neuronal IAHP was fully suppressed upon application of benzamil 30 µM (104 ± 3% inhibition, n=11), whilst recombinant SK(1-3) currents in human embryonic kidney (HEK) cells were inhibited by 45%, 26% and 34% (n=6, 10 and 7) respectively at the same concentration under symmetric [K+] conditions (IC50 35-67 µM). The inhibition of SK channels in HEK cells persisted in a physiological K+ concentration gradient, and was stronger at negative voltages. Whilst the different extent of inhibition in the two systems hints at additional actions of the drug in neurons, the suppression of SK channels by benzamil corroborates our findings on the modulation of Ca2+ signals by SK channels and points at this and other amiloride analogues as potential lead compounds for the development of selective and reversible SK channel inhibitors. The results also prompt a careful reassessment of the effects of benzamil on Ca2+ transients in native systems, given the spectrum of ion channels and exchangers this compound targets within a similar range of concentrations.