The purpose of this study was to determine the subcellular site(s) of the renal cortical phospholipidosis induced by aminoglycosides. For this purpose we injected male Sprague-Dawley rats s.c. with netilmicin, containing tracer quantities of [3H]netilmicin, at 100 mg/kg/day for 2 days; control rats were injected with saline. Twenty-four hours after the second injection of drug the rats were sacrificed and the renal cortex was fractionated by differential ultracentrifugation and Percoll gradient density techniques to obtain purified lysosomes, mitochondria, microsomes, brush border membranes and basolateral membranes. The total phospholipid content of the renal cortex was 300 +/- 5 nmol/mg of protein in control rats and 340 +/- 5 nmol/mg of protein in netilmicin-injected rats. The total phospholipid content of the lysosomal fraction of netilmicin rats, which was enriched in myeloid bodies and [3H]netilmicin, was 91% greater than that of control rats and reflected significant increases of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol. This pattern is identical to that reported previously for the rat renal cortical phospholipidosis induced by aminoglycosides. The total phospholipid contents of the mitochondrial, microsomal, brush border membrane and basolateral membrane fractions of netilmicin-injected rats were higher by approximately 10% than the respective fractions of control rats and each fraction exhibited a significant increase of one or more of the four phospholipids elevated in the renal cortical homogenate and in the lysosomal fraction. The data indicate that the myeloid body is the primary source of the lysosomal phospholipidosis induced by netilmicin which provides support for the hypothesis that the lysosomal phospholipidosis is secondary to aminoglycoside-induced inhibition of phospholipid degradation. In addition the findings of increased phospholipid content and altered phospholipid composition of the other subcellular fractions raise the possibility that aminoglycoside antibiotics cause a more generalized disturbance of phospholipid metabolism characterized by altered synthesis as well as degradation in renal proximal tubular cells.