Measurements of intracellular and luminal ion activities as well as membrane potential were used to calculate electrochemical gradients for Cl–, Na+, K+ and H+ across the apical membrane during fluid secretion by Malpighian tubules of Rhodnius prolixus. The results show that the contribution of Na+/H+ and/or K+/H+ exchangers to fluid secretion is feasible both in unstimulated and serotonin-stimulated tubules. Similarly, the electrochemical potential for Cl– is consistent with the passive movement of Cl– from cell to lumen through Cl– channels. The contribution of apical K+:Cl– cotransport and/or paracellular Cl– movement to net transepithelial ion transport is thermodynamically unfeasible. pH in the lumen (pH 6.08±0.1, N=6) was more acid than in the bath (pH 7.25±0.01, N=26) and serotonin stimulation produced a significant increase in lumen pH to 6.32±0.04 (N=5). Intracellular pH was 6.97±0.01 and 6.82±0.04 in unstimulated and serotonin-stimulated tubules, respectively. Lumen pH was altered whereas intracellular pH was tightly regulated during serotonin and bumetanide treatment. Furthermore, DIDS or amiloride treatment did not affect intracellular pH. However, intracellular pH shifted 0.25 pH units more acid in Na+-free saline, suggesting that a Na+-dependent pH regulatory mechanism is at play in steady state pH regulation during fluid secretion by Malpighian tubules of Rhodnius prolixus. The data are consistent with a role for a basolateral Na+/H+ exchanger in intracellular pH regulation during fluid secretion.