1.Buffer conditions in in vitro metabolism studies using human liver microsomes (HLM) have been reported to affect the metabolic activities of several cytochrome P450 (CYP) isozymes in different ways, although there are no reports about the dependence of CYP2C8 activity on buffer conditions.2.The present study investigated the effect of buffer components (phosphate or Tris-HCl) and their concentration (10-200mM) on the CYP2C8 and CYP3A4 activities of HLM, using paclitaxel and triazolam, respectively, as marker substrates.3.The K-m (or S-50) and V-max values for both paclitaxel 6-hydroxylation and triazolam - and 4-hydroxylation, estimated by fitting analyses based on the Michaelis-Menten or Hill equation, greatly depended on the buffer components and their concentration.4.The CLint values in phosphate buffer were 1.2-3.0-fold (paclitaxel) or 3.1-6.4-fold (triazolam) higher than in Tris-HCl buffer at 50-100mM. These values also depended on the buffer concentration, with a maximum 2.3-fold difference observed between 50 and 100mM which are both commonly used in drug metabolism studies.5.These findings suggest the necessity for optimization of the buffer conditions in the quantitative evaluation of metabolic clearances, such as in vitro-in vivo extrapolation and also estimating the contribution of a particular enzyme in drug metabolism.