This study was aimed to investigate the pathogenesis of toluene-induced renal tubular acidosis (RTA). In 5 individuals addicted to toluene sniffing we documented the occurrence of hypokalemia and hyperchloremic metabolic acidosis associated with inability to lower urine pH below 5.5 (6.06 ± 0.24). Overall kidney bicarbonate reabsorption was normal or enhanced, a feature characteristic of the distal form of RTA (DRTA). These findings resemble those found during the administration of amphotericin B, a drug felt to cause DRTA by increasing hydrogen ion (H<sup>+</sup>) back-diffusion in the collecting tubule. In toluene sniffers, the urine pCO<sub>2</sub> measured in a highly alkaline urine was reduced (47 ± 8.8 mm Hg), suggesting a decrease in the rate of collecting tubule H<sup>+</sup> secretion rather than H<sup>+</sup> back-diffusion. To investigate these two mechanisms of altered distal acidification more directly we studied the effect of toluene on acidification by the urinary turtle bladder, an epithelial analogue of the mammalian collecting tubule. In this preparation, toluene resulted in a decrease in the rate of H<sup>+</sup> secretion measured by either the pH stat technique or the reverse short circuit current. When mucosal pH was progressively lowered to examine H<sup>+</sup> secretion against an H<sup>+</sup> gradient, toluene-treated bladders displayed a significant decrease in proton conductance but the lowest mucosal pH required to nullify H<sup>+</sup> secretion, (MpH) JH = O, was not different from that of control bladders (4.05 ± 0.29 and 3.90 ± 0.13, respectively). In contrast, in amphotericin B-treated bladders (MpH) JH =·was 5.15 ± 0.39, a value more than 1 pH unit higher than that of control and toluene-treated bladders (p < 0.05). Thus, amphotericin B, but not toluene, reduced the pH gradient that could be generated across the turtle bladder. These findings suggest that toluene, unlike amphotericin B, does not cause H<sup>+</sup> back-diffusion. Decreased conductance of protons through the active transport pathway is the mechanism that best explains the toluene-induced defect in distal acidification.