Aflatoxin B(1)-lysine adduct (AFB-Lys) is a reliable biomarker for aflatoxin exposure; however, a systematic toxicokinetic evaluation has not been reported. In this study, male F344 rats were orally exposed to single, or repeated, doses of AFB(1) and the toxicokinetics of serum AFB-Lys that followed treatments were investigated. A single-dose of AFB(1) increased serum AFB-Lys levels rapidly peaking at 4h, followed by first-order elimination, through which the half-life was estimated to be 2.31 days. A physiologically based pharmacokinetic model showed that approximately 3.00-3.90% and 1.12-1.98% of the administered AFB(1) doses were converted to serum AFB-Lys adducts at 2h and 24h post treatment, respectively. Repeated AFB(1) exposure at 5-25 μg/kg body weight linearly increased serum AFB-Lys levels for 5 weeks in animals, resulting in a 1-1.5 times higher AFB-Lys level overall. This indicates the potential of this adduct as a reliable biomarker for repeated low dose exposure. Higher dose exposure at 75 μg/kg increased the level of AFB-Lys to a maximum at 2 weeks, followed by a gradual decrease to near plateau level up to 5 weeks. In conclusion, this study systematically evaluated the toxicokinetics of serum AFB-Lys adduct in F344 rats using a physiologically based pharmacokinetic model and robust statistical modeling analysis and provided a firm and clear understanding of the toxicokinetics of this biomarker.