The hepatic carcinogen aflatoxin B1 (AFB1) is metabolized in the liver by at least four different P450s, all of which exhibit large interindividual differences in the expression levels. These differences could affect the individual risk of hepatocellular carcinoma (HCC). We investigated the metabolism of AFB1 in a panel of 13 human liver microsomal preparations using a hepatic abundance model, which takes into account the specific kinetic parameters and the expression levels of these P450s. We found a 12-fold variability in the production rate of the carcinogenic metabolite AFB1-8,9-epoxide (AFBO) and a 22-fold variability in the production of the detoxification product AFQ1. The ratio between the AFBO and the AFQ1 production rates varied between 1:19 and 1:1.7. P450 3A4 contributed a majority of AFBO and AFQ1, and its expression level was the most important determinant of the AFB1 disposition toward these primary metabolites. P450 3A5, which exclusively produced AFBO, was the second-most important enzyme activating AFB1 to AFBO, followed by P450 3A7 and P450 1A2. The relative contribution of AFBO by P450 3A5 strongly depended on the concomitant expression of P450 3A4, and it was as high as 15% in a P450 3A5 high expressor with the lowest P450 3A4 expression of all livers. The P450 1A2-specific AFB1 detoxification product AFM1 was not detected. In conclusion, the variable expression of P450s has a major effect on the carcinogenic activation of AFB1, which may affect the individual predisposition to HCC. P450 3A4 expression is the most important determinant of AFB1 activation to AFBO. The contribution of P450 1A2 to AFB1 metabolism appears to be negligible and may have been overestimated. Targeted chemoprevention of AFB1-associated HCC should consider P450 3A4 inhibitors and avoidance of P450 3A4 inducers.