The time course of contrast media (CM)-induced renal proximal tubular vacuolation was investigated in rats by light microscopy, transmission electron microscopy (TEM), and ultrastructural histochemistry for acid phosphate activity. Young adult male rats were treated with a single dose of 3.0 g I/kg Iotrolan (Isovist 300 mg I/ml) and sacrificed at 0 min, 5-min, 15-min, 15-min, 2-hr, and 24-hr intervals. Light microscopy of vibratome sections of freshly excised tissue of cryostat and paraffin sections was also performed to allow comparison of the appearance of the vacuoles in the fresh state with light and electron microscopy. The sequence of events seen to occur can be summarized as follows. CM-induced vacuolation occurred at a low level a soon as 5 min after compound administration. The vacuolation was observed by TEM but could not be detected by light microscopy. This was followed by an increase in size and numbers of vacuoles up to the 24-hr timepoint with a sequential increase in the staining for acid phosphatase activity of the vacuoles, most marked at the 24-hr timepoint. At timepoints less than 24 hr there appeared to be no marked increased in the normal complement by lysosomes or in the components of the Golgi-endoplasmic reticulum-lysosome pathway. At 24 hr, the vast majority, but not all, of the CM-induced vacuoles were positive for acid phosphatase activity. The intensity of staining varied, and there was evidence of infusion of small lysosomes with CM-induced vacuoles. These results suggest that formation of CM-induced vacuoles is a 2-stage process, following a normal pathway for the handling of endogenous and exogenous substances.