In brush-border membrane vesicles from rabbit ileum, we previously reported pH gradient-stimulated SO4 uptake and presented evidence that this represents carrier-mediated SO4-OH exchange. In the present study inhibitors of SO4-OH exchange (H-SO4 cotransport) were shown not to inhibit Na-SO4 cotransport, suggesting that these are two separate carrier-mediated transport mechanisms. While pH gradient-stimulated SO4 uptake was inhibited 87% by 0.1 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, disodium salt (DIDS) and 79% by 1.0 mM furosemide, Na+-stimulated SO4 uptake was only inhibited 11 and 0%, respectively. K+ (20 mM), Cl (5 mM), and oxalate (0.25 mM) inhibited pH gradient-stimulated SO4 uptake (38-65%) but had no effect on Na+-stimulated SO4 uptake. Finally, at Na+ concentrations (10 mM) significantly less than that required for Na+-stimulated SO4 uptake (60-100 mM), external Na+ inhibited pH gradient-stimulated SO4 uptake, suggesting two independent effects of this cation. SO4 uptake was also inhibited by external K+ both in the presence and absence of a pH gradient. A Dixon plot of the DIDS-sensitive SO4 uptake under pH gradient conditions yielded a straight line, indicating a single site of interaction between external K+ and the SO4-OH carrier (apparent Ki = 7.2 mM). In contrast to the inhibition by external K+, internal K+ stimulated SO4 uptake. This effect was DIDS sensitive and not enhanced by valinomycin, suggesting an interaction of internal K+ with the SO4-OH exchanger independent of a K+-induced electrical potential. SO4 uptake and the effects of K+ were pH modulated with less SO4 uptake and less K+ effect at higher pH.(ABSTRACT TRUNCATED AT 250 WORDS)