Selection of in vitro models that accurately characterize metabolite systemic and hepatobiliary exposure remains a challenge in drug development. In the present study, mechanisms underlying differences in systemic exposure of two active metabolites, furamidine and 2,5-bis (5-amidino)-2-pyridyl furan (CPD-0801), were examined using two hepatic models from rats: isolated perfused livers (IPLs) and sandwich-cultured hepatocytes (SCH). Pafuramidine, a prodrug of furamidine, and 2,5-bis [5-(N-methoxyamidino)-2-pyridyl] furan (CPD-0868), a prodrug of CPD-0801, were selected for investigation because CPD-0801 exhibits greater systemic exposure than furamidine, despite remarkable structural similarity between these two active metabolites. In both IPLs and SCH, the extent of conversion of CPD-0868 to CPD-0801 was consistently higher than that of pafuramidine to furamidine over time (at most 2.5-fold); area under the curve (AUC) of CPD-0801 in IPL perfusate and SCH medium was at least 7-fold higher than that of furamidine. Pharmacokinetic modeling revealed that the rate constant for basolateral (liver to blood) net efflux (k(A_net efflux)) of total formed CPD-0801 (bound + unbound) was 6-fold higher than that of furamidine. Hepatic accumulation of both active metabolites was extensive (>95% of total formed); the hepatic unbound fraction (f(u,L)) of CPD-0801 was 5-fold higher than that of furamidine (1.6 versus 0.3%). Incorporation of f(u,L) into the pharmacokinetic model resulted in comparable k(A_net efflux,u) between furamidine and CPD-0801. In conclusion, intrahepatic binding markedly influenced the disposition of these active metabolites. A higher f(u,L) explained, in part, the enhanced perfusate AUC of CPD-0801 compared with furamidine in IPLs. SCH predicted the disposition of prodrug/metabolite in IPLs.