Single cell calcium microfluorimetry was used to examine the regulation of [Ca 2+] i homeostasis in a clonal cell line of corticotropes (AtT-20 cells). Single cells, loaded with fura-2/AM, were exposed briefly to elevated potassium chloride (KCl, 40 mM, 5 sec). The time constant of decay of the [Ca 2+] i signal was used as an index of [Ca 2+] i extrusion and/or sequestration. Substitution of extracellular sodium with lithium, N-methyl-D-glucamine (NMDG), or Tris, increased resting levels of [Ca 2+] i and significantly increased the time constant of [Ca 2+] i decay by 40% compared to control indicating the participation of Na +-Ca 2+-exchange. Prior exposure of single cells to thapsigargin (1 μM) or BuBHQ (10 μM), inhibitors of the SERCA Ca 2+-ATPases, and/or the mitochondrial uncoupler FCCP (1 μM) did not significantly change the time constant of [Ca 2+] i decay following KCl. Lanthanum ions (La +3), applied during the decay of the KCl-induced increase in [Ca 2+] i, significantly increased the time constant of the return of [Ca 2+] i to resting levels by 70% compared to control. Brief exposure of cells to sodium orthovanadate, an inhibitor of ATP-dependent pump activity, slowed and longer exposures prevented, the return of [Ca 2+] i to resting levels. We conclude that neither intracellular SERCA pumps nor mitochondrial uptake contribute significantly to [Ca 2+] i sequestration following a [Ca 2+] i load and that the plasma membrane Ca 2+-ATPase contributes to a greater extent than the Na +-Ca 2+-exchanger to the return of [Ca 2+] i to resting levels following a [Ca 2+] i load under these experimental conditions.