Polychlorinated Biphenyls (PCBs) are environmental contaminants that are associated with adverse human health effects, including cancer and neurotoxicity. PCBs are metabolized by cytochrome P450 (P450) enzymes in the liver to hydroxylated metabolites (HO-PCBs) that are potentially more toxic than the parent PCBs. Hydroxylated PCBs are further metabolized to PCB glucuronide and/or sulfate conjugates, which are excreted through urine and feces. In this project, we hypothesize that the knockout of specific P450 isoforms affects the urinary excretion of PCB 136 and its metabolites in mice. To test this hypothesis, Cyp1a1 and Cyp1a2 knockout mice and congenic wildtype mice were exposed to a single dose of PCB 136 (30 mg/kg body weight). Pooled urine samples were collected daily for 3 days after exposure. Levels of PCB 136 and four metabolites, including 2,2′,3,3′,6,6′-hexachlorobiphenyl-5-ol (5-OH PCB 136), 2,2′,3,3′,6,6′-hexachlorobiphenyl-4-ol (4-OH PCB 136), 2,2′,3,3′,6,6′-hexachlorobiphenyl-4,5-diol (4,5-diOH PCB 136) and 2,2′,3,4′,6,6′-hexachlorobiphenyl-3′-ol (3′-OH PCB 150) were measured after deconjugation with β-glucuronidase/sulfatase using gas chromatography with electron capture detection. PCB 136, 5-OH PCB 136, 4-OH-PCB 136 and 4,5-diOH PCB 136 were detected in all urine samples. Urinary levels of PCB 136 and its metabolites decreased from day 1 to day 3. Profiles of OH-PCBs showed some differences based on genotype. These results demonstrate that the knockout of individual P450 isoforms affects the urinary elimination of PCB metabolites, a fact that needs to be considered when studying the toxicity of PCBs in transgenic animals, such as Cyp1a1 and Cyp1a2 knockout mice.