The opossum kidney (OK) line displays PTH-mediated activation of adenylyl cyclase and phospholipase C and inhibition of phosphate (Pi) uptake via regulation of the type IIa sodium-phosphate cotransporter, consistent with effects in vivo. OKH cells, a subclone of the OK cell line, robustly activates PTH-mediated activation of adenylyl cyclase, but is defective in PTH-mediated inhibition of sodium-phosphate cotransport and signaling via phospholipase C. Compared with wild-type OK cells, OKH cells express low levels of the Na+/H+ exchanger regulatory factor 1 (NHERF-1). Stable expression of NHERF-1 in OKH cells (OKH-N1) rescues the PTH-mediated inhibition of sodium-phosphate cotransport. NHERF-1 also restores the capacity of 8-bromo-cAMP and forskolin to inhibit Pi uptake, but the PTH dose-response for cAMP accumulation and inhibition of Pi uptake differ by 2 orders of magnitude. NHERF-1, in addition, modestly restores phorbol ester-mediated inhibition of Pi uptake, which is much weaker than that elicited by PTH. A poor correlation exists between the inhibition of Pi uptake mediated by PTH ( approximately 60%) and the inhibition mediated by phorbol 12-myristate 13-acetate ( approximately 30%) and the ability of these molecules to activate the protein kinase C-responsive reporter gene. Furthermore, we show that NHERF-1 directly interacts with type IIa cotransporter in OK cells. Although, PTH-mediated inhibition of Pi uptake in OK cells is largely NHERF-1 dependent, the signaling pathway(s) by which this occurs is still unclear. These pathways may involve cooperativity between cAMP- and protein kinase C-dependent pathways or activation/inhibition of an unrecognized NHERF-1-dependent pathway(s).