The effect of phosphorylated phloretins on Na⁺-dependent phosphate uptake into rabbit renal brush border membrane vesicles (BBMV) was examined. Na⁺-dependent phosphate uptake into isolated rabbit cortex BBMV was sensitive to 2'-phosphophloretin (2'-PP) and 2'-phospho-4',4,6'-trimethoxy phloretin (PTMP) in a dose-dependent and pH-dependent manner. PTMP inhibition of Na⁺-dependent phosphate uptake was maximum at alkali pH and 2'-PP inhibition of Na⁺-dependent phosphate uptake was maximum at acidic pH. Increasing Na⁺ concentrations did not increase PTMP inhibition of renal cortex BBMV Na⁺-dependent phosphate uptake at pH 6. The effect of phosphophloretins on Na⁺-dependent phosphate uptake was examined in BBMV isolated from purified proximal tubules, and distal tubules. 2'-PP and PTMP inhibition of Na⁺-dependent phosphate uptake into BBMV isolated from purified proximal tubules was similar to the inhibition seen with BBMV from renal cortex. 2'-PP, but not PTMP inhibited Na⁺-dependent phosphate uptake into BBMV isolated from purified distal tubules. The pH dependence of inhibition, the absence of PTMP inhibition of Na⁺-dependent phosphate uptake into distal tubule BBMV, and the inhibition of Na⁺- dependent phosphate uptake into distal tubule BBMV suggest that NaPi Ia is 2'-PP-sensitive, and NaPi II a is PTMP-sensitive.