The effect of gentamicin exposure was investigated in LLC-PK1 cells in culture. Gentamicin (0.5-2.0 mM) was added to the medium of cells which had been grown to confluency in the absence of antibiotics and antimycotics. Exposure to gentamicin (1-4 days) did not effect total cellular protein or DNA levels, total cell number or the release of various marker enzymes to the medium. ATP levels in gentamicin-treated cells did not differ from control cells; however, medium from the gentamicin-treated cells contained significantly lower lactic acid levels. Morphological examination by electron microscopy revealed gentamicin-elicited myeloid body formation. Furthermore, total phospholipid level was elevated markedly in gentamicin-treated cells. Analysis of specific phospholipid classes showed only phosphatidylcholine, phosphatidylinositol and polyphosphoinositide phospholipid levels increased in a time-dependent manner. Phosphatidylinositol showed the highest percentage of increase. Raising the normal medium calcium concentration (0.2 mg/ml) 1.5-, 2.0- or 3.0-fold did not alter gentamicin-induced elevation in cellular phosphatidylinositol and phosphatidylcholine. Gentamicin exposure also resulted in a concentration-dependent increase in the turnover of LLC-PK1 cell-free fatty acids, monoglyceride, diglyceride and nonesterified cholesterol and a decrease in triglyceride turnover. Calcium transport into and through the cell monolayer was inhibited markedly by gentamicin despite the fact that 45Ca++ binding to gentamicin-treated cells was greater. These results demonstrate that manifestations of gentamicin toxicity in LLC-PK1 cells parallel those reported in the whole animal thus making the LLC-PK1 cell in culture a valid system for elucidating the mechanism of gentamicin-elicited alterations in renal epithelium.