AbstractPidepletion of proximal tubule cells isolated from mouse kidney results in a decrease in the cell content of fructose‐2,6‐bisphosphate and an increase in the rate of gluconeogenesis from pyruvate, malate and succinate. Gluconeogenesis from glycerol is unaffected by Pidepletion. Introduction of fructose‐2,6‐bisphosphate into the cytosol of ATP‐permeabilized cells is accompanied by a fall in gluconeogenesis.The presence of external Ca2+stimulates gluconeogenesis. When cytosolic Ca2+is raised to 1·8 μM by permeabilization, the resealed cells still require 2·5 mM Ca2+in the bathing medium in order to perform gluconeogenesis at the maximum rate. Cells permeabilized in the presence of cAMP show a decreased rate of glucose production. Phorbol ester stimulates gluconeogenesis provided that the phorbol treatment is performed in the absence of Ca2+ions.It is suggested that Pidepletion may stimulate pyruvate carboxylase activity and facilitate the entry of certain gluconeogenic substrates into mitochondria. It is also proposed that important aspects of the control of renal gluconeogenesis by parathyroid hormone are mediated by prot