Recently, we reported that aristolochic acid (AA) a naturally occurring nephrotoxin and carcinogen is implicated in a unique type of renal fibrosis, designated Chinese herbs nephropathy (CHN). Indeed, we identified the principal aristolochic acid-DNA adduct in the kidney of five such patients. We now extend these observations and demonstrate the presence of additional AA-DNA adducts by the 32P-post-labelling method not only in the kidneys, but also in a ureter obtained after renal transplantation. Using the nuclease P1 version of the assay not only the major DNA adduct of aristolochic acid, 7-(deoxyadenosin-N6-yl)-aristolactam I (dA-AAI), but also the minor adducts, 7-(deoxyguanosin-N2-yl)-aristolactam I (dG-AAI) and 7-(deoxyadenosin-N6-yl)-aristolactam II (dA-AAII) were detected, and identified by cochromatographic analyses with TLC and HPLC. Quantitative analyses of six kidneys revealed relative adduct levels from 0.7 to 5.3/10(7) for dA-AAI, from 0.02 to 0.12/10(7) for dG-AAI and 0.06 to 0.24/ 10(7) nucleotides for dA-AAII. The detection of the dA-AAII adduct is consistent with the occurrence of aristolochic acid II (AAII) in the herb powder imported under the name of Stephania tetrandra and confirms that the patients had indeed ingested the natural mixture of AAI and AAII. 32P-post-labelling analyses of further biopsy samples of one patient showed the known adduct pattern of AA exposure not only in the kidney, but also in the ureter, whereas in skin and muscle tissue no adduct spots were detectable. In an attempt to explain the higher level of the dA-AAI adduct compared to the dG-AAI adduct level in renal tissue even 44 months after the end of regimen, the persistence of these two purine adducts was investigated in the kidney of rats given a single oral dose of pure AAI. In contrast to the dG-AAI adduct, the dA-AAI adduct exhibited a lifelong persistence in the kidney of rats. Our data demonstrate that AA forms DNA adducts in human tissue by the same activation mechanism(s) reported from animal studies. Thus, the carcinogenic/mutagenic activity of AA observed in animals could also be responsible for the urothelial cancers observed in two of the CHN patients.