The functional significance of the apical vacuolar-type proton pump (V-ATPase) in Drosophila Malpighian tubules was studied by measuring the intracellular pH (pHi) and luminal pH (pHJ with double-barrelled pH-microelectrodes in proximal segments of the larval anterior tubule immersed in nominally bicarbonate-free solutions (pH o 6.9). In proximal segments both pH i (7.43 4-0.20) and pHlu (7.10 ___ 0.24)) were significantly lower than in distal segments (pH i 7.70_+0.29, pH]u 8.09+0.15). Steady-state pH i of proximal segments was much less sensitive to changes in pH o than pH of the luminal fluid (ApH1u/ApH o was 0.49 while ApHi/ApH o was 0.18; pH o 6.50-7.20). Re-alkaliniziation from an NH4Cl-induced intracellular acid load (initial pH i recovery rate 0.55 ___ 0.34 pH.min 2) was nearly totally inhibited by 1 mmol,1-1 KCN (96% inhibition) and to a large degree (79%) by 1 btmol-1-1 bafilomycin Av In contrast, both vanadate (1 mmol.1-1) and amiloride (1 mmol.1 1) inhibited pH i recovery by 38% and 33%, respectively. Unlike amiloride, removal of Na + from the bathing saline had no effect on pHi recovery, indicating that a Na+/ H + exchange is not significantly involved in pHi regulation. Instead pH i regulation apparently depended largely on the availability of ATP and on the activity of the bafilomycin-sensitive proton pump.