The potent inhibitory activity of novel 2-benzyltetralone and 2-benzylidenetetralone derivatives vs liver microsomal retinoic acid metabolizing enzymes and a MCF-7 CYP26A1 cell assay is described. In the liver microsomal assay, the 2-biphenylmethyl-6-hydroxytetralone derivatives 16a and 16b were found to be potent inhibitors (IC50 = 0.5 and 0.8 microM) compared with the broad spectrum P450 inhibitor ketoconazole and the retinoid mimetic R115866 (IC50 = 18.0 and 9.0 microM, respectively). In the MCF-7 CYP26A1 cell assay, the 2-(4-hydroxybenzyl)-6-methoxytetralone 5 and unsaturated benzylidene precursor 6 were found to be the most potent (IC50 = 7 and 5 microM, respectively), which was comparable with liarozole (7 microM) but considerably less active than R115866 (IC50 = 5 nM). With a CYP26A1 homology model, the tetralones were shown to be positioned in a hydrophobic tunnel with additional interactions, e.g., transition metal coordination and hydrogen-bonding interactions with GLY300, observed for the potent 4-hydroxyphenyl substituted inhibitors.