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Aflatoxin B1 (AFB1), one of the most common mycotoxins found in human foods and animal feed, is principally hepatotoxic and hepatocarcinogenic. The aim of the present study was to explore the effect of AFB1 on messenger RNA (mRNA) expression of pregnane X receptor (PXR), constitutive androstane receptor (CAR), and aryl hydrocarbon receptor (AhR) and some of their target cytochromes using primary cultures of human hepatocytes. Our results showed that AFB1, at noncytotoxic increasing concentrations, caused a significant upregulation of cytochrome P 2B6 (CYP2B6), CYP3A5, and to a lesser extent CYP3A4 and CYP2C9. Pregnane X receptor and CAR mRNA expression increased in the 3 treated livers. Aflatoxin B1 was found also to induce an overexpression of CYP1A1 and CYP1A2 genes accompanied by an increase in AhR mRNA expression. These findings suggest that AFB1 could activate PXR, CAR, and AhR; however, further investigations are needed to confirm nuclear receptor activation by AFB1.