A study was conducted to identify the cytochrome P450 (CYP, CYP450) enzyme orthologs involved in the bioactivation of aflatoxin B1 (AFB1) into the highly toxic metabolite known as aflatoxin-8,9-epoxide (AFBO) in quail and chicken hepatic microsomes. The strategies used included the use of specific CYP450 inhibitors and the correlation of prototype substrate activities with AFBO production. Additionally, the presence of the enzymes was qualitatively determined using an immunoblotting technique. The results showed that both quail and chicken microsomes have CYP1A1, CYP1A2, CYP2A6, and CYP3A4 enzymatic activity. A strong relationship between CYP1A1 and CYP2A6 activities and AFB1 bioactivation was found in both species. Inhibition studies provided more evidence for the role of CYP2A6 in the bioactivation of AFB1. The immunoblot results showed clear bands for the CYP2A6 and CYP3A4 orthologs in both species. The results of the present study indicate that CYP2A6 and, to a lesser extent, CYP1A1 are responsible for the bioactivation of AFB1 into AFBO in both quail and chicken hepatic microsomes.