Both insulin deficiency and glycosuria are known to inhibit the tubular reabsorption of phosphate. This inhibition has previously been evaluated either in the fasted state or on a normal phosphate diet. The goal of this study was to evaluate how dietary phosphate depletion affected the relative effects of insulin deficiency and glycosuria on the tubular reabsorption of phosphate. Rats were maintained on either a low- (0.03%) or normal (0.8%) phosphate diet. After 5 days, one half of the animals in each group received streptozotocin to induce short-term insulin deficiency, whereas the other half received vehicle alone. Two days later, sodium-dependent phosphate uptake by renal brush border membrane vesicles (BBMV) was evaluated in each of the four experimental groups. The effect of glucose on phosphate uptake was determined by the addition of varying concentrations of glucose (between 0 and 32 mmol/L) to the extravesicular transport fluid. BBMV phosphate uptake was about threefold higher in the nondiabetic rats fed a low-phosphate diet as compared with the nondiabetic animals maintained on a normal phosphate diet. In rats maintained on a low-phosphate diet, streptozotocin treatment prevented the increase in BBMV phosphate transport; in contrast, in animals fed a normal phosphate diet, streptozotocin treatment had no effect on BBMV phosphate transport. Extravesicular glucose significantly inhibited phosphate transport in a dose-related manner, regardless of dietary phosphate or insulin status. Because fasting mimics the catabolic state associated with insulin deficiency, BBMV phosphate transport was also measured in rats fasted for 48 h after the administration of streptozotocin or vehicle. In these fasted rats, streptozotocin treatment had no effect on phosphate uptake, whereas extravesicular glucose inhibited phosphate transport in both treatment groups. These observations suggest that the effects of insulin deficiency and glycosuria on phosphate uptake by the proximal tubule BBMV differ according to the dietary regimen.