The efficacy and distribution of soluble and insoluble tetracyclines were studied in rats with experimental pyelonephritis due to E. coli. Insoluble oxytetracycline (IOT) was noted to persist in normal kidneys in concentration sufficient to inhibit bacterial growth for as long as 96 h after its injection. Similarly, this preparation significantly prevented the development of pyelonephritis in the rat when administered as long as 96 h prior to bacterial inoculation. Fine crystals of insoluble tetracycline (ITC) were then prepared by alkalinization of a solution of commercially available soluble tetracycline (TC). A single injection of ITC was markedly superior to a single injection of TC in treatment of experimental pyelonephritis in rats, and equivalent to a 6-day course of the latter. Labelled (7–3H) soluble and insoluble tetracyclines were then prepared and their concentrations after i. m. and i. v. injection compared by liquid scintillation counting of selected tissues of normal and pyelonephritic rats (i. e. normal kidney, pyelonephritic lesions, plasma, fat, lung, liver, heart, and spleen). Although TC concentrations were higher from 4 to 12 h after drug injection, at 24 h kidney and plasma concentrations of TC and ITC were equivalent, and by 48 and 96 h the ITC concentrations in the plasma and kidney were significantly greater. Both compounds displayed higher levels in the kidney than in other tissues, with the exception of the site of i. m. injection, or the lung after i. v. injection, where high concentrations of ITC suggested that the insoluble form of the drug formed a depot. In contrast to Castenada’s original suggestion that the insoluble antibiotic was more effective because of phagocytosis of particles of the antibiotic which were then brought to the site of infection, the current data suggest that this is due to depot formation followed by preferential concentration in the kidney, as compared to other tissues. These data indicate that mere changes in the physical property of tetracyclines thus can alter their effectiveness in experimental pyelonephritis.