Aims: Success of the quantitative prediction of drug-drug interactions via inhibition of CYP-mediated metabolism from the inhibitor concentration at the enzyme active site ([I]) and the in vitro inhibition constant (Ki) is variable. The aim of this study was to examine the impact of the fraction of victim drug metabolized by a particular CYP (fmCYP) and the inhibitor absorption rate constant (ka) on prediction accuracy. Methods: Drug-drug interaction studies involving inhibition of CYP2C9, CYP2D6 and CYP3A4 (n = 115) were investigated. Data on fmCYP for the probe substrates of each enzyme and ka values for the inhibitors were incorporated into in vivo predictions, alone or in combination, using either the maximum hepatic input or the average systemic plasma concentration as a surrogate for [I]. The success of prediction (AUC ratio predicted within twofold of in vivo value) was compared using nominal values of fmCYP = 1 and K a = 0.1 min-1. Results: The incorporation of f mCYP values into in vivo predictions using the hepatic input plasma concentration resulted in 84% of studies within twofold of in vivo value. The effect of ka values alone significantly reduced the number of over-predictions for CYP2D6 and CYP3A4; however, less precision was observed compared with the fmCYP. The incorporation of both fmCYP and ka values resulted in 81% of studies within twofold of in vivo value. Conclusions: The incorporation of substrate and inhibitor-related information, namely fmCYP and ka, markedly improved prediction of 115 interaction studies with CYP2C9, CYP2D6 and CYP3A4 in comparison with [I]/Ki ratio alone. © 2005 Blackwell Publishing Ltd.