14C-Benzidine (BZ) was added to the recirculating perfusate of the isolated perfused rat liver. The system was monitored at timed intervals for the disappearance of BZ and the appearance of metabolites. BZ was extensively metabolized by this system and after 2 hr of perfusion greater than 95% of the administered radiolabel was in the form of metabolic products. In the perfusate the concentration of BZ declined rapidly whereas the concentration of N-acetyl-BZ (ABZ) increased temporarily and then declined. The concentration of N,N'-diacetyl-BZ (DABZ) increased with time and by 1 hr DABZ had become the major metabolite in the system. In the bile, which contained 22% of the dose after 2 hr, BZ-N-glucuronide and ABZ-glucuronide were the major metabolites initially, but after 1 hr of perfusion N-hydroxy-DABZ-glucuronide had become the major biliary metabolite. Addition of BZ and 35S-Na2SO4 to the perfusate resulted in at least one 35S-containing metabolite. Other major metabolites excreted in bile included 3-hydroxy-DABZ glucuronide, ABZ, and DABZ. DABZ underwent deacetylation to ABZ and N-hydroxy-DABZ underwent rapid reduction to DABZ when added to the isolated liver system. Qualitatively similar biliary metabolite patterns at later times were observed when either BZ, DABZ, or N-hydroxy-DABZ was added to the perfusate.