As a result of the widespread use of Cd in industry and its extensive dissemination in the environment, there has been considerable interest in the identification of early biomarkers of Cd-induced kidney injury. Kim-1 is a transmembrane glycoprotein that is not detectable in normal kidney, but is up-regulated and shed into the urine following ischemic or nephrotoxic injury. Recent studies utilizing a sub-chronic model of Cd exposure in the rat have shown that Kim-1 is an early urinary marker of Cd-induced kidney injury. Kim-1 was detected in the urine 4-5 weeks before the onset of proteinuria and 1-3 weeks before the appearance of urinary metallothionein and Clara cell protein 16, which are standard markers of Cd nephrotoxicity. In the present study, we have compared the time course for the appearance of Kim-1 in the urine with the time course for the appearance of alpha glutathione-S-transferase (alpha-GST), N-acetyl-beta-D-glucose amidase (NAG) and Cd, each of which have been used or proposed as urinary markers of Cd nephrotoxicity. Adult male Sprague-Dawley rats were given daily subcutaneous injections of 0.6 mg (5.36 micromoles)/kg Cd, 5 days per week for up to 12 weeks. One day each week, 24 h urine samples were collected and analyzed for protein, creatinine and the various markers. The results showed that significant levels of Kim-1 appeared in the urine as early as 6 weeks into the treatment protocol and then continued to rise for the remainder of the 12 week treatment period. By contrast, significant levels of alpha-GST and NAG did not appear in the urine until 8 and 12 weeks, respectively, while proteinuria was not evident until 10 weeks. The urinary excretion of Cd was below the level of detection until week 4 and then showed a slow, linear increase over the next 6 weeks before increasing markedly between weeks 10 and 12. These results provide additional evidence that Kim-1 is a sensitive biomarker of the early stages of Cd-induced proximal tubule injury.