Glycerophosphocholine (GPC) is an osmoprotective compatible and counteracting organic osmolyte that accumulates in renal inner medullary cells in response to high NaCl and urea. We previously found that high NaCl increases GPC in renal [Madin-Darby canine kidney (MDCK)] cells. The GPC is derived from phosphatidylcholine, catalyzed by a phospholipase that was not identified at that time. Neuropathy target esterase (NTE) was recently shown to be a phospholipase B that catalyzes production of GPC from phosphatidylcholine. The purpose of the present study was to test whether NTE contributes to the high NaCl-induced increase of GPC synthesis in renal cells. We find that in mouse inner medullary collecting duct cells, high NaCl increases NTE mRNA within 8 h and NTE protein within 16 h. Diisopropyl fluorophosphate, which inhibits NTE esterase activity, reduces GPC accumulation, as does an siRNA that specifically reduces NTE protein abundance. The 20-h half-life of NTE mRNA is unaffected by high NaCl. TonEBP/OREBP is a transcription factor that is activated by high NaCl. Knockdown of TonEBP/OREBP by a specific siRNA inhibits the high NaCl-induced increase of NTE mRNA. Further, the lower renal inner medullary interstitial NaCl concentration that occurs chronically in ClCK1-/- mice and acutely in normal mice given furosemide is associated with lower NTE mRNA and protein. We conclude that high NaCl increases transcription of NTE, likely mediated by TonEBP/OREBP, and that the resultant increase of NTE expression contributes to increased production and accumulation of GPC in mammalian renal cells in tissue culture and in vivo.