GDP-sensitive Cl- uniport is a widely studied property of the uncoupling protein of brown adipose tissue mitochondria; nevertheless, little is known about its mechanism and there is even controversy over whether this protein transports Cl-. Using a fluorescent probe assay, we have demonstrated non-ohmic, electrophoretic, GDP-sensitive Cl- uniport into proteoliposomes reconstituted with purified uncoupler protein. We have also identified a large number of new anionic substrates for this porter that also inhibit Cl- uniport competitively. Anion transport, its inhibition by GDP and anion inhibition of Cl- uniport are all strongly dependent on anion hydrophobicity. These surprising results are consequential for hypotheses of common transport mechanisms in the gene family of mitochondrial anion porters.