This laboratory has studied a number of flavone derivatives for aryl hydrocarbon receptor (AhR) agonist and antagonist potential using cell-free and cell culture systems. The current report extends these investigations by testing the potent AhR antagonist 3'-methoxy-4'-nitroflavone (3'M4'NF) for in vivo activity. Wild-type C57Bl/6 male mice were treated with solvent, benzo[a]pyrene (B[a]P; 150 mg/kg), or concurrently with B[a]P and 3'M4'NF (60 mg/kg; delivered as a split dose). Since B[a]P is bioactivated to genotoxic metabolites by AhR-regulated enzymes, we measured B[a]P-induced chromosomal damage in peripheral blood (i.e. micronuclei) to characterize the antagonistic potential of 3'M4'NF in vivo. The influence of AhR signal transduction was investigated further by challenging wild-type and Ahr null allele mice with B[a]P with and without a 3'M4'NF co-treatment. The micronucleus data obtained from these experiments indicated that 3'M4'NF can attenuate the genotoxicity of B[a]P significantly. Since 3'M4'NF also protected Ahr null allele mice from B[a]P-induced genetic damage, it was apparent that AhR-independent mechanisms contribute to the effects observed. However, as opposed to the protective effects observed with the micronucleus endpoint, histological observations and lethality data indicated that some B[a]P effects are enhanced by 3'M4'NF. Potentiated B[a]P toxicity may be explained by inhibition of basal and induced CYP1A1/2 activities. Both in vitro and in vivo data presented herein support this hypothesis.