Consumption of foods contaminated with aflatoxin B₁ (AFB₁) is a recognized risk factor for developing hepatocellular carcinomas (HCCs). The mutational signature of AFB₁ is characterized by high frequency G > T transversions in a limited subset of trinucleotide sequences. The 8,9-dihydro-8-(2,6-diamino-4-oxo-3,4-dihydropyrimid-5-yl-formamido)-9-hydroxyaflatoxin B₁ (AFB₁-FapyGua) has been implicated as the primary DNA lesion responsible for AFB₁-induced mutations. This study evaluated the mutagenic potential of AFB₁-FapyGua in four contexts, including hot- and cold-spot sequences as apparent in the mutational signature. Vectors containing AFB₁-FapyGua were replicated in primate cells and the products of replication were isolated and sequenced. Regardless of the sequence context, AFB₁-FapyGua caused base substitutions at frequencies of ~ 80-90%, with G > T transversions being most common. Spectra of mutations were only slightly modulated by the sequence context. These data suggest that mechanism(s) defining sequence context-dependent distribution of AFB₁-induced mutations likely operates prior to replication.