To investigate the inhibitory effect of aza-polycyclic aromatic compounds on cytochrome P450 (CYP) 2C9 activity and analyze the fluorine-substitution effects on the CYP2C9 inhibition, benzo[h]quinoline (BhQ) and its fluorinated derivatives, 3-F-, 5-F-, 6-F-, 9-F-, 10-F-, 3,6-diF-, 5,6-diF-, 7,10-diF-BhQ, were subjected to analysis of their inhibitory effects on recombinant human CYP2C9.1-catalyzed tolbutamide hydroxylation. Although the inhibitory activity of BhQ itself on tolbutamide hydroxylation was not very strong (IC₅₀ = 157 μ M), the inhibitory activities of BhQ derivatives on tolbutamide hydroxylation varied with the substituted fluorine positions. Their inhibitory activities decreased in the following order; (BhQ) > 7,10-diF ≥ 3,6-diF, 9-F ≥ 10-F > 6-F ≥ 5-F, 3-F ≥ 5,6-diF-BhQ. Moreover, the fluorine-substitution effect on the inhibition of tolbutamide hydroxylation catalyzed by wild-type CYP2C9 (CYP2C9.1) was different from the effects on the hydroxylation catalyzed by its polymorphic isozymes CYP2C9.2 and CYP2C9.3. The inhibitory activities of BhQs on tolbutamide hydroxylation by CYP2C9.2 decreased in the following order; 5-F ≥ 10-F, 9-F ≥ (BhQ) ≥ 7,10-diF > 6-F ≥ 5,6-diF, 3,6-diF > 3-F-BhQ, and those on the hydroxylation by CYP2C9.3 decreased in the follwing order; 7,10-diF, 10-F, (BhQ) ≥ 9-F, 5-F > 3-F ≥ 5,6-diF, 6-F, 3,6-diF-BhQ. The results thus showed that the position-specific substitution by fluorine atom(s) altered the CYP2C9 inhibition by BhQ derivatives in different manners depending on the polymorphic isozymes involved. These results suggest that inhibitory profiles obtained with fluorine-substituted analogs of the key inhibitor molecule may be useful as a new tool for phenotyping the polymorphic CYP isoforms.