Sulfate uptake in apical membrane vesicles isolated from bovine tracheal epithelium is shown to occur into an osmotically sensitive intravesicular space, via a carrier-mediated systen. This conclusion is based on three lines of evidence: 1) saturation kinetics; 2) substrate specificity; and 3) inhibition by the anion transport inhibitors SITS and DIDS. The affinity of the transport system is highest in low ionic strength media (apparent K(m) = 0.13 mM) and decreases in the presence of gluconate (apparent K(m) = 0.68 mM). Chloride appears to cis-inhibit sulfate uptake and to trans-stimulate sulfate efflux. Cis-inhibition and trans-stimulation studies with a variety of anions indicate that this exchange system may be shared by HCO3-, S2O32-, SeO42-, and MoO42- but not by H2PO4- or HAsO42-. Studies indicate that protons may play two distinct roles in sulfate transport in this system. 1) Their possible modifier role is suggested by the fact that protons affect SO42- transport in an uncompetitive manner. 2) The possibility that the proton gradient may act as an energy source for a secondary active transport is indicated by the fact that the imposition of a proton gradient stimulates a transient movement of sulfate into the tracheal apical membrane vesicle, against its concentration gradient, causing an 'overshoot' phenomenon. Our studies show that the carrier-mediated system can function in the absence of chloride. The overshoot observed in the presence of a proton gradient (OH- gradient) indicates that under those conditions the mechanism of transport may be a SO42--OH- exchange. The fact that chloride cis-inhibits and trans-stimulates SO42- transport indicates that SO42- uptake may also occur via a SO42--Cl- exchange. Studies carried out so far do not enable us to conclude unequivocally whether the tracheal apical membrane system displays two distinct carrier activities (SO42--Cl-;SO42--OH-) or one anion exchanger, which like the erythrocyte anion exchanger, may interact with SO42-, Cl-, and H+. The fact that anion transport inhibitors DIDS and SITS inhibit SO42- transport in the presence or absence of chloride suggests that the latter possibility may be the case.