Effects of d-limonene on alpha 2u-globulin in the kidneys, urine and serum were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting analysis. Treatment of male rats with d-limonene by gavage for 14 consecutive days (300 mg/kg/day) caused accumulation of hyaline droplets in renal proximal tubule cells, and a marked intensification of a protein band corresponding to the kidney-type alpha 2u-globulin, with a molecular weight calculated to be approximately 16 kDa. However, no significant changes in the serum alpha 2u-globulin (native-type) band, of approximately 19 kDa, were observed between treated rats and controls, suggesting that circulating alpha 2u-globulin levels were not affected by the d-limonene administration. While the molecular weight of the major alpha 2u-globulin in the urine from control rats was the same as that in the serum (native-type), marked increase in the protein band corresponding to kidney-type-alpha 2u-globulin was observed in the urine from treated rats. The results were indicative of elimination of alpha 2u-globulin from the kidney to urine, the appearance of kidney-type-alpha 2u-globulin in urine implying disruption or exfoliation of proximal tubule cells. Therefore, it is suggested that the presence of the alpha 2u-globulin (kidney-type) in the urine might be used as an indicator of chemically induced alpha 2u-globulin nephropathy.