Intracellular ion activities (a(ion)) and basolateral membrane potential (Vbl) were measured in Malpighian tubule cells of Rhodnius prolixus using double-barrelled ion-selective microelectrodes. In saline containing 103 mmol l(-1) Na+, 6 mmol l(-1) K+ and 93 mmol l(-1) Cl-, intracellular ion activities in unstimulated upper Malpighian tubules were 21, 86 and 32 mmol l(-1), respectively. In serotonin-stimulated tubules, aCl was unchanged, whereas aNa increased to 33 mmol l(-1) and aK declined to 71 mmol l(-1). Vbl was -59 mV and -63 mV for unstimulated and stimulated tubules, respectively. Calculated electrochemical potentials (deltamuF) favour passive movement of Na+ into the cell and passive movement of Cl- out of the cell in both unstimulated and serotonin-stimulated tubules. Passive movement of K+ out of the cell is favoured in unstimulated tubules. In stimulated tubules, deltamuF for K+ is close to 0 mV. The thermodynamic feasibilities of Na+-K+-2Cl-, Na+-Cl- and K+-Cl- cotransporters were evaluated by calculating the net electrochemical potential (deltamu(net)/F) for each transporter. Our results show that a Na+-K+-2Cl- or a Na+-Cl- cotransporter but not a K+-Cl- cotransporter would permit the movement of ions into the cell in stimulated tubules. The effects of Ba2+ and ouabain on Vbl and rates of fluid and ion secretion show that net entry of K+ through ion channels or the Na+/K+-ATPase can be ruled out in stimulated tubules. Maintenance of intracellular Cl- activity was dependent upon the presence of both Na+ and K+ in the bathing saline. Bumetanide reduced the fluxes of both Na+ and K+. Taken together, the results support the involvement of a basolateral Na+-K+-2Cl- cotransporter in serotonin-stimulated fluid secretion by Rhodnius prolixus Malpighian tubules.