A cidification of the luminal solution by the isolated turtle bladder involves H⁺ secretion by a pump at the luminal membrane. The OH⁻ dissociated in this process reacts with CO₂ and forms HCO₃⁻ which moves passively out of the cell across the serosal cell membrane. In the present study, this exit step for HCO₃⁻ was inhibited by serosal addition of the disulfonic stilbene, SITS, an agent which is thought to bind to a transport protein at the serosal cell membrane. 90 min after serosal addition of 0.5 mM SITS, H⁺ secretion decreased by > 80%. In contrast, luminal addition of SITS had no effect. During inhibition of H⁺ secretion by serosal SITS, overall cell pH, measured by the 5, 5-dimethyl-2, 3-oxazolidinedione method, increased from 7.48±0.03 to 7.61±0.02. This increase of 0.13±0.02 pH U was associated with a much larger regional pH increase as judged from the decrement in the attainable pH gradient across the epithelium. After serosal SITS, this gradient was reduced from 2.88±0.06 to 2.09±0.11 pH U. In the absence of evidence for increased H⁺ permeability or a change in the force of the H⁺ pump, the gradient decrement of 0.79±0.08 U reflects a similar pH increment on the cytoplasmic side of the pump.SITS inhibits the exit of bicarbonate across the serosal cell membrane and, thereby, creates a compartment of high alkalinity in series with the pump. The increased electrochemical gradient across the active transport pathway is the primary factor in the inhibition of urinary acidification.