Abstract Glucose 6-phosphatase from rat liver and kidney microsomes previously has been shown to catalyze an inorganic pyrophosphate-glucose phosphotransferase reaction. Nordlie and Soodsma recently have suggested that formation and then degradation of glucose 6-phosphate by the combined action of phosphotransferase and phosphohydrolase activities of this enzyme may under certain conditions constitute part of a mechanism for facilitated transport of glucose across the kidney tubular cell. To attempt to gain additional insight regarding the possible significance of such a physiological role for the enzyme, studies of the effect of phlorizin, a potent inhibitor of tubular sugar transport, on both phosphohydrolase and phosphotransferase activities of this enzyme were undertaken. Inhibition, in vitro, of both glucose 6-phosphate phosphohydrolase and pyrophosphate-glucose phosphotransferase activities of kidney homogenates and microsomal suspensions prepared in isotonic sucrose solution was noted over the entire range of phlorizin concentrations studied, 6.7 x 10-5 m to 6.7 mm. Sensitivity of activities to inhibition by this compound was either partially or completely abolished by supplementation of the homogenates or microsomal suspensions, to 0.2% (w/v), with sodium deoxycholate, sodium cholate, digitonin, Triton X-100, or Tween 20. Phlorizin was without discernible effect on activity of partially purified enzyme preparations obtained by fractional ammonium sulfate precipitation of deoxycholate-dispersed microsomal suspensions. Inhibition of phosphotransferase activity in homogenates or microsomes was markedly potentiated by the cationic detergent trimethylhexadecylammonium bromide. Kinetic analyses indicated that inhibition of both phosphohydrolase and phosphotransferase activities by phlorizin was of the classical noncompetitive type. A value of approximately 0.7 mm for Ki for phlorizin was determined in the absence of detergent; the value was reduced to 0.2 InM when trimethylhexadecylammonium bromide was added (to 0.2%, w/v) to enzyme preparations prior to assay for activity. The effects of the administration in vivo of phlorizin on phosphotransferase activity of the enzyme were also studied. Reductions in activity per mg of kidney protein of as much as 18% were produced by intravenous injection of 0.1-g doses of phlorizin to rats. The data obtained appear to rationalize the initially apparently contradictory reports of earlier workers regarding the effects (or lack of effect) of phlorizin on glucose 6-phosphatase. The experimental observations reported in this paper also are interpreted to constitute added support for, but not direct proof of, the possible involvement of synthetic and hydrolytic activities of this enzyme in kidney glucose transport. It is suggested that the modifying effects of various detergents on phlorizin-sensitivity of the enzyme are manifest through selective action on both active and inactive silent phlorizin-binding sites in the enzyme preparation.