Tamoxifen has been reported to inhibit acidification of cytoplasmic organelles in mammalian cells. Here, the mechanism of this inhibition is investigated using in vitro assays on isolated organelles and liposomes. Tamoxifen inhibited ATP-dependent acidification in organelles from a variety of sources, including isolated microsomes from mammalian cells, vacuoles from Saccharomyces cerevisiae, and inverted membrane vesicles from Escherichia coli. Tamoxifen increased the ATPase activity of the vacuolar proton ATPase but decreased the membrane potential (Vm) generated by this proton pump, suggesting that tamoxifen may act by increasing proton permeability. In liposomes, tamoxifen increased the rate of pH dissipation. Studies comparing the effect of tamoxifen on pH gradients using different salt conditions and with other known ionophores suggest that tamoxifen affects transmembrane pH through two independent mechanisms. First, as a lipophilic weak base, it partitions into acidic vesicles, resulting in rapid neutralization. Second, it mediates coupled, electroneutral transport of proton or hydroxide with chloride. An understanding of the biochemical mechanism(s) for the effects of tamoxifen that are independent of the estrogen receptor could contribute to predicting side effects of tamoxifen and in designing screens to select for estrogen-receptor antagonists without these side effects.