The midgut epithelium of Drosophila has recently attracted attention on two fronts: the identification of regenerative cells (1-3) with a developmental plan analogous to mammalian gastrointestinal crypts, and identification in both larval and adult forms of at least three architecturally distinct segments, two of which transport acid and base (4,5). The larval posterior midgut is particularly powerful in secreting base into the lumen where the pH can exceed 10 in the steady state. This is achieved by extrusion of H⁺ across the basal membrane of the epithelium into the haemolymph by an electrogenic H⁺ V-ATPase, located in or very close to the basal membrane (4,5). The epithelium is tight, and the basal membrane is ≈ 8-fold more resistive than the apical, enabling voltage-clamp of this tubular epithelium to study the steady-state extrusion of H⁺ as a function of individual cell membrane potential. The epithelium was cannulated at both ends, perfused as described (4) with control Ringer solution and connected to electrometers for measurement of transepithelial parameters. The extracellular solution was scanned orthogonal to the basement membrane with a liquid ion-exchanger pH microelectrode to detect the pH profile adjacent to the basement membrane(pH_o); intracellular pH (pH_i), corrected for membrane potential, was also measured. Current was injected into the perfusion end (potential V_p) of the tubule with a bridge-balance electrometer and the small attenuation of the voltage at the collection end (V_c) was used to calculate transepithelial resistance (R_t) and short-circuit current (I_sc) using terminated cable analysis. Paired data±SEM are given below. In control Ringer the lumen-negative V_t was -35.6 ± 1.1mV, R_t was 1167± 85 Ω.cm² and I_sc was 34.41 ± 1.9 µA.cm^-2 (n=34). Supplementation of the control Ringer with a 2:1 mixture of Ringer and Schneider insect medium (Sigma) prolonged viability, increased V_t to -45.4 ± 1.3mV, decreased R_t to 847 ± 43 Ω.cm², increased I_sc to 58.5 ± 3.4 µA.cm^-2 (n=34, p<0.01) and increased the resistance ratios of the basal to apical membranes from 5.23 ± 1.0 to 21.5 ± 4.3 (n=8, p<0.01). Injection of -120, -200 and -300 nA currents to the perfusion end hyperpolarized V_c by -21 ± 8, -33 ± 6, -54 ± 5mV respectively and increased pH_o by 0.15 ± 0.01 (n=3), 0.38 ± 0.04 (n=5) and 0.14 ± 0.03 (n=5) units respectively. Injection of 180 ± 40nA (n=3) hyperpolarized V_c by -34 ± 6mV and increased pH_i by 0.34 ± 0.14 units (n=3). We conclude that the hyperpolarization of V_t reduces the H⁺ efflux by the H⁺ V-ATPase (increase of pH_o) and also increases backflux into the lumen through non-selective channels in the apical membrane (as evidenced by increase in pH_i).