Rat thymocytes incorporate large amounts of acridine orange at concentrations of approximately 10 pM in about 10 minutes at 37OC. The addition of (NH4)2S04 (at a final concentration of several rnMolars) releases about 30% of the incorporated dye into t h e medium. The NH4+-releasable dye uptake is almost completely abolished by 20 minutes' incubation with 4 pM of the Ca+ +-ionophore A23187. Dye uptake is associated with an absorbance change at 492 m m and thus may be followed spectrophotometrically. However, NH4+ at the above concentrations or nigericin (0.5 mg/ml) completely annul this change in absorbance, indicating that it reflects only the accumulation of the dye within t h e acidic cellular compartments. In a Ca+ +-containing physiological saline, the addition of A23187 (at a final concentration of 8 pM) at the end of t h e dye uptake phase initiated a reversal in the absorbance change; in the absence of Ca+ +, reversal occurred at a much lower rate. Incubation of cells for 30 minutes with 2 pM A23187 in Ca+ +-containing saline completely abolished NH,+-sensitive dye uptake; less A23187 (0.5 pM) and like or a longer incubation period brought about a striking decrease in NH4+-sensitive dye uptake. Similar results were obtained with cells suspended in RPLM 1640 medium. In the absence of external C a + + , A23187 impaired cell capacity to incorporate dye in a ApH-dependent manner, but a longer incubation time or higher concentrations of t h e ionophore were required to obtain a comparable effect. It is thus concluded that the ionophore dissipates intracellular pH gradients by an intracellular divalent cation (Ca+ + or also Mg+ +)-H+ exchange.