A variety of chemicals, including triacetyloleandomycin (TAO), alpha-naphthoflavone (ANF), and diethyldithiocarbamate (DDC), are widely used as inhibitory probes for select individual human cytochrome P450 (CYP) enzymes, despite the fact that the selectivity of these inhibitors has not been rigorously evaluated. In the present study we take advantage of recent advances in cDNA-directed human P450 expression to evaluate directly the P450 form selectivity of TAO, ANF, and DDC, using a panel of 10 individual cDNA-expressed human P450s. Under experimental conditions known to yield maximal TAO complexation with P450 hemoproteins, TAO (20 microM) inhibited the catalytic activity of expressed CYPs 3A3, 3A4, and 3A5, whereas it did not affect CYPs 1A1, 1A2, 2A6, 2B6, 2C8, 2C9, or 2E1 activity. ANF inhibited not only CYPs 1A1 and 1A2 (IC50 = 0.4-0.5 microM), but it was also similarly effective against CYPs 2C8 and 2C9. Increasing the concentration of ANF to 10 microM led to inhibition of CYP2A6 and CYP2B6. Although a previous study suggested that DDC is a selective inhibitor of CYP2E1, the present investigation shows that at concentrations required to inhibit CYP2E1 (IC50 approximately 125 microM when preincubated with NADPH), DDC also inhibited CYPs 1A1, 1A2, 2A6, 2B6, 2C8, 3A3, and 3A4. Decreasing the concentration of DDC to 10 microM, however, led to inhibition of CYP2A6 (65% inhibition) and CYP2B6 (50% inhibition), but none of the other P450s examined, including CYP2E1. Overall, these results establish that (a) TAO is a selective inhibitor of the human CYP3A subfamily; (b) ANF potently inhibits CYP2C8 and CYP2C9, in addition to CYPs 1A1 and 1A2; and (c) DDC cannot be employed as a diagnostic inhibitory probe for CYP2E1.