The effect of ketoconazole, a CYP3A inhibitor, on the oral bioavailability of tirilazad mesylate was assessed in 12 healthy subjects, who received the following treatments in a crossover design: a) 10 mg/kg tirilazad mesylate solution orally on the fourth day of a 7-day regimen of 200 mg ketoconazole once daily, b) 10 mg/kg tirilazad mesylate solution orally, c) 2 mg/kg i.v. tirilazad mesylate solution on the fourth day of a 7-day regimen of 200 mg ketoconazole once daily and d) 2mg/kg i.v. tirilazad mesylate solution. Plasma concentrations of tirilazad mesylate and its active reduced metabolites (U-89678 and U-87999) were measured by high-performance liquid chromatography. Urinary ratios of 6 beta-hydroxycortisol to cortisol (6 beta-OHC/C) were measured as an index of hepatic CYP3A activity. Ketoconazole increased mean tirilazad mesylate area under the curve (AUC) values by 67% and 309% for i.v. and oral administration, respectively. Mean AUC values for U-89678 were increased 472% and 720% by ketoconazole coadministration with i.v. and oral tirilazad, respectively, whereas increases of > 10-fold in mean U-87999 AUC values were observed. These differences were statistically significant. These results indicate that ketoconazole inhibits the metabolism of these three compounds, which suggests that all of the compounds are substrates for CYP3A. Urinary 6 beta-OHC/C ratios did not reflect this level of effect of ketoconazole on CYP3A; this probe may not be useful for assessing the effect of CYP3A inhibitors. The absolute bioavailability of oral tirilazad was 8.7 +/- 4.8%; ketoconazole increased the bioavailability to 20.9 +/- 6.5%. Ketoconazole increased tirilazad mesylate bioavailability by decreasing the first-pass liver and gut wall metabolism of tirilazad mesylate to similar degrees.