Mitochondrial volume regulation depends on K + movement across the inner membrane and a mitochondrial Ca 2+ -dependent K + channel (mitoK Ca ) reportedly contributes to mitochondrial K + uniporter activity. Here we utilize a novel K Ca channel activator, NS11021, to examine the role of mitoK Ca in regulating mitochondrial function by measuring K + flux, membrane potential (ΔΨ m ), light scattering, and respiration in guinea pig heart mitochondria. K + uptake and the influence of anions were assessed in mitochondria loaded with the K + sensor PBFI by adding either the chloride (KCl), acetate (KAc), or phosphate (KH 2 PO 4 ) salts of K + to energized mitochondria in a sucrose-based medium. K + fluxes saturated at ∼10 mM for each salt, attaining maximal rates of 172±17, 54±2.4, and 33±3.8 nmol K + /min/mg in KCl, KAc, or KH 2 PO 4 , respectively. NS11021 (50 nM) increased the maximal K + uptake rate by 2.5-fold in the presence of KH 2 PO 4 or KAc and increased mitochondrial volume, with little effect on ΔΨ m . In KCl, NS11021 increased K + uptake by only 30% and did not increase volume. The effects of NS11021 on K + uptake were inhibited by the K Ca toxins charybdotoxin (200 nM) or paxilline (1 μM). Fifty nanomolar of NS11021 increased the mitochondrial respiratory control ratio (RCR) in KH 2 PO 4 , but not in KCl; however, above 1 μM, NS11021 decreased RCR and depolarized ΔΨ m . A control compound lacking K Ca activator properties did not increase K + uptake or volume but had similar nonspecific (toxin-insensitive) effects at high concentrations. The results indicate that activating K + flux through mitoK Ca mediates a beneficial effect on energetics that depends on mitochondrial swelling with maintained ΔΨ m .