Mechanisms of ion transport during primary urine formation by the Malpighian tubule of Drosophila melanogaster were analyzed through measurements of fluid secretion rate, transepithelial ion flux, basolateral membrane potential (Vbl) and intracellular activities of K+ (aKi) and Cl–(aCli). Calculation of the electrochemical potentials for both ions permitted assessment of the possible contributions of K+ channels, Na+:K+:2Cl–cotransport, and K+:Cl– cotransport, to net transepithelial ion secretion across the basolateral membrane. The data show that passive movement of both K+ and Cl– from cell to bath is favoured across the basolateral membrane, indicating that both ions are actively transported into the cell. Contributions of basolateral K+ channels or K+:Cl– cotransporters to net transepithelial ion secretion can be ruled out. After prior exposure of tubules to ouabain, subsequent addition of bumetanide reduced fluid secretion rate, K+ flux and Na+ flux, indicating a role for a Na+:K+:2Cl– cotransporter in fluid secretion. Addition of the K+ channel blocker Ba2+ had no effect on aKi or aCli. Addition of Ba2+ unmasked a basolateral Cl– conductance and the hyperpolarization of Vbl in response to Ba2+ was Cl–-dependent. A new model for fluid secretion proposes that K+ and Cl– cross the basolateral membrane through a Na+-driven Na+:K+:2Cl–cotransporter and that most of the Na+ that enters the cells is returned to the bath through the Na+/K+-ATPase.