In mice treated with 5-aminolevulinic acid (ALA) and polyhalogenated aromatic compounds, the levels of both hepatic cytochrome P450 (CYP)1A2 and iron—which can be quite different among inbred strains—are critical in causing experimental uroporphyria. Here we investigate the development of uroporphyria as a function of CYP1A2 and iron levels in the liver of mice having a common C57BL/6 genetic background. We compared Cyp1a2(−/−) knockout mice, Cyp1a2(+/−) heterozygotes, Cyp1a2(+/+) wild type, and Cyp1a2(+/+) mice pretreated with a low dose of 3,3',4,4',5-pentachlorobiphenyl (PCB126) (4 μg/kg). Cyp1a2(+/−) mice contain about 60% of the hepatic CYP1A2 content of Cyp1a2(+/+) mice, and the PCB126-pretreated Cyp1a2(+/+) mice have about twice the wild-type levels of CYP1A2. ALA- and iron-treated Cyp1a2(+/+) mice are known to accumulate hepatic uroporphyrin; this accumulation was increased 7-fold by pretreatment with the low dose of PCB126. ALA- and iron-treated Cyp1a2(+/−) heterozygote mice accumulated no uroporphyrin in 4 weeks, but by 8 weeks accumulated significant amounts of uroporphyrin. As previously reported, the ALA- and iron-treated Cyp1a2(−/−) knockout mouse has no CYP1A2 and exhibits no detectable uroporphyrin accumulation. Iron dose-response curves in ALA- and PCB126-treated Cyp1a2(+/+) mice showed that hepatic iron levels greater than 850 μg/g liver were required to produce significant uroporphyrin accumulation in the liver. Other measures of hepatic effects of iron (iron-response element-binding protein [IRP]-iron response element [IRE] binding activity and accumulation of protoporphyrin from ALA) decreased when the level of iron was considerably lower than 850 μg/g liver. At low iron doses, accumulation of iron was principally in Kupffer cells, whereas at the higher doses (required to stimulate uroporphyrin accumulation), more iron was found in parenchymal cells. We conclude that small changes in hepatic CYP1A2 levels can dramatically affect uroporphyria in C57BL/6 mice, providing the animals have been sufficiently loaded with iron; these data might be clinically relevant to acquired (sporadic) porphyria cutanea tarda, because humans show greater than 60-fold genetic differences in hepatic basal CYP1A2.