The phosphorylation of the sodium-hydrogen exchanger regulatory factor-1 (NHERF-1) plays a key role in the regulation of renal phosphate transport by parathyroid hormone (PTH) and dopamine. Ser(77) in the first PDZ domain of NHERF-1 is a downstream target of both hormones. The current experiments explore the role of Thr(95), another phosphate acceptor site in the PDZ I domain, on hormone-mediated regulation of phosphate transport in the proximal tubule of the kidney. The substitution of alanine for threonine at position 95 (T95A) significantly decreased the rate and extent of in vitro phosphorylation of Ser(77) by PKC. In NHERF-1-null proximal tubule cells, neither PTH nor dopamine inhibited sodium-dependent phosphate transport. Infection of the cells with adenovirus expressing full-length WT GFP-NHERF-1 increased basal phosphate transport and restored the inhibitory effect of both PTH and dopamine. Infection with full-length NHERF-1 containing a T95A mutation, however, increased basal phosphate transport but not the responsiveness to either hormone. As determined by surface plasmon resonance, the substitution of serine for aspartic acid (S77D) in the PDZ I domain decreased the binding affinity to the sodium-dependent phosphate transporter 2a (Npt2a) as compared with WT PDZ I, but a T95D mutation had no effect on binding. Finally, cellular studies indicated that both PTH and dopamine treatment increased the phosphorylation of Thr(95). These studies indicate a remarkable cooperativity between the phosphorylation of Thr(95) and Ser(77) of NHERF-1 in the hormonal regulation of renal phosphate transport. The phosphorylation of Thr(95) facilitates the phosphorylation of Ser(77). This, in turn, results in the dissociation of NHERF-1 from Npt2a and a decrease in phosphate transport in renal proximal tubule cells.