A Na+ gradient (extravesicular greater than intravesicular) increased the rate of inorganic sulfate (SO24-) uptake into renal brush-border membrane vesicles and energized the transient accumulation of the anion against its concentration gradient, indicating a secondary active transport system. Stimulation of SO24- uptake was specific for Na+. The anions, SO23-, S2O23-, SeO24-, MoO24-, CrO24-, and WO24-, but not HPO24-, cis-inhibited and trans-stimulated SO24- uptake, suggesting that these divalent anions shared the SO24- carrier. The Na+/SO24- co-transport and Na+. The apparent Km for SO24- was 0.6 mM at 100 mM Na+. The relationship between Na+ concentration and rate of SO24- uptake was sigmoidal. From a Hill analysis of the data a [Na+]0.5 of 36 mM and an n value of 1.6 were calculated. Comparisons of the effects of a K+ diffusion potential (inside positive), of a H+ diffusion potential (inside negative), of Na+ salts of anions of different conductances on the Na+-dependent uptakes of SO24- and D-glucose, and of the responses of a membrane potential-sensitive fluorescent probe concomitant with the uptakes indicate that Na+/SO24- co-transport was electroneutral. The simplest stoichiometry consistent with an electroneutral mechanism would be the co-transport of two Na+ and one SO24-. Na+ gradient-dependent SO24- uptake was enhanced by intravesicular Cl-. cis-Cl- inhibited the efflux as well as the influx of SO24-. These findings suggest that Cl- was an inhibitor of SO24- transport. Intravesicular K+ stimulated Na+ gradient-dependent SO24- uptake. The co-transport of Na+/SO24- appeared not to be coupled to the transmembrane flux of K+. It is hypothesized that the co-transport system contained an internal site activated by K+.