The present study was undertaken to examine the interaction of phosphonoformic acid (PFA) with the Na(+)-sulfate cotransporter and the effect of thyroid hormone (triiodothyronine; T3) on Na(+)-dependent sulfate transport and Na(+)-dependent PFA binding in mouse renal brush-border membrane vesicles. PFA inhibits Na(+)-dependent sulfate transport in a competitive manner [apparent inhibitory constant (Ki) = 4.3 +/- 1.1 mM]. T3 administered in pharmacological doses significantly stimulates Na(+)-dependent sulfate transport in renal brush-border membranes compared with vehicle-treated controls. Although T3 has no effect on Na(+)-dependent glucose transport, T3 also stimulates Na(+)-dependent phosphate transport. Kinetic studies demonstrate that T3 increases the apparent maximal velocity (Vmax) for Na(+)-sulfate cotransport without changing the apparent Michaelis constant (Km). T3 does not significantly affect either Na(+)-dependent PFA binding or the phosphate- and sulfate-displaceable components of Na(+)-dependent PFA binding. Finally, Na(+)-dependent brush-border membrane sulfate transport is unchanged in phosphate-deprived mice that exhibit increased Na(+)-phosphate cotransport and in X-linked Hyp mice that exhibit impaired Na(+)-phosphate cotransport. The present results demonstrate that 1) PFA is a competitive inhibitor of Na(+)-sulfate cotransport, 2) T3 stimulates Na(+)-dependent sulfate, as well as Na(+)-dependent phosphate transport, but has no effect on PFA binding, and 3) phosphate deprivation and the X-linked Hyp mutation do not influence Na(+)-sulfate cotransport.(ABSTRACT TRUNCATED AT 250 WORDS)