To determine the sites of tubular phosphate reabsorption in the nephron, microinjection studies were undertaken, utilizing isotonic electrolyte solutions, containing either 1.4 or 8.0 mM phosphate and radioactive PO(4)-(33)P and inulin-(3)H, in rats made mildly diuretic by infusion of mannitol. The injected sites were localized by the technique of latex dissection. The relation between proximal tubular length and per cent (33)P recovery for injections of 1.4 mM phosphate (physiological amounts) suggest that relatively little reabsorption of phosphate occurs in the distal 30% of the proximal tubule compared with the proximal portion of the tubule. The corresponding recoveries for proximal tubular microinjections of 8.0 mM phosphate fall along a smooth curve tending to plateau with essentially complete (33)P recovery (> 95%) beyond 50% of the tubule. Absolute reabsorption of injected phosphate for both concentrations (i.e., absolute efflux per unit tubular length in the proximal tubule) was independent of phosphate delivery, since the relationship between reabsorption and site of injection was no different for the two concentrations. Distal convoluted tubular microinjections for both phosphate concentrations showed complete recovery of (33)P from all injection sites. THE DATA INDICATE THAT: (a) no phosphate reabsorption occurs in the distal convoluted tubule or in the collecting duct, (b) phosphate efflux per unit tubular length is greater in the first one-third of the proximal tubule than in the remaining two-thirds, and (c) in the last two-thirds of the proximal tubule, absolute phosphate reabsorption is relatively small and might be limited by factors other than the amount or concentration of injected phosphate.