Gentamicin induces a phospholipiduria in the rat. We sought to determine whether the phospholipiduria reflected shedding of brush border membrane by measuring the urinary excretion of membrane marker enzymes, by defining the composition of the urinary phospholipids, and by examining the renal cortex and urinary sediment using transmission electron microscopy of rats injected with gentamicin sulfate (100 mg/kg of body weight for 1 to 6 days). The results were compared and contrasted with those of rats injected with mercuric chloride, a nephrotoxin known to cause selective injury to the brush border membrane. Gentamicin-injected rats exhibited a phospholipiduria that by the third day was 10-fold above the baseline level. In contrast the brush border membrane enzymes alanine aminopeptidase and gamma-glutamyl transferase and the lysosomal enzyme N-acetyl-beta-glucosaminidase increased less than 1-fold above baseline. Electron microscopy of the renal cortex revealed the presence of myeloid bodies within lysosomes of proximal tubule cells and lying free within the lumen. The brush border membrane was largely intact. The urine sediment was dominated by the presence of myeloid bodies; there was little evidence of brush border membrane fragments. The urinary phospholpids were enriched in phosphatidylinositiol, phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. This pattern is similar to that of a lysosomal fraction enriched in myeloid bodies. Rats injected with mercuric chloride exhibited a phospholipiduria associated with sharp increases (7- to 15-fold) in the urinary excretion of brush border membrane enzymes, whereas N-acetyl-beta-glucosaminidase increased less than 3-fold. Electron microscopy revealed blebbing of the apical membrane with loss of microvilli of proximal tubular cells particularly evident along the pars recta. The urinary sediment contained microvilli and fragments of plasma membrane but no myeloid bodies. The urine contained large quantities of sphingomyelin. In the rat renal cortex the brush border membrane is the major source of this phospholipid. We conclude that the phospholipiduria induced by mercuric chloride is derived primarily from the shedding of brush border membrane fragments into the urine. In contrast, the urinary excretion of phospholipid induced by gentamicin reflects primarily the extrusion of the lysosomal myeloid body from proximal tubular cells. We speculate that the origin of the phospholipids making up the lysosomal myeloid body may be the endocytic vesicle that mediates the transport of aminoglycosides into the lysosomal system.