Cytochrome P450s are a superfamily of monooxygenases that catalyze numerous stereo- and regio-specific oxygenation reactions that are potentially valuable biotransformations. In particular, plant P450s typically have higher substrate specificity than mammalian P450s. Since most eukaryotic P450s are membrane-bound proteins, require the cofactor NAD(P)H and cytochrome P450 reductase (CPR), a whole-cell system is preferred for application in biosynthesis. Cinnamate 4-hydroxylase (C4H) is a P450 monooxygenase from the phenylpropanoid pathway, which catalyzes the hydroxylation of cinnamic acid with high specificity. In this study, recombinant Saccharomyces cerevisiae co-expressing C4H and CPR from Arabidopsis thaliana was selected as a system to develop an optimal medium for the heterologous expression of plant P450s. A high throughput screening (HTS) method based on the natural substrate was developed to examine factors important for whole cell C4H activity, which included the ratio of inducer (galactose) to glucose and concentration of casein hydrolysate. A single-stage procedure that combined cell growth and induction was optimized through factorial design to simplify cell culture and enzyme expression. (c) 2005 Elsevier Inc. All rights reserved.