Pairs of forward and reverse primers and TaqMan probes specific to each of 52 human phase I metabolizing enzymes (alcohol dehydrogenase, aldehyde dehydrogenase, aldehyde oxidase, dihydropyrimidine dehydrogenase, epoxide hydrolase, esterase, flavin-containing monooxygenase, monoamine oxidase, prostaglandin endoperoxide synthase, quinone oxidoreductase, and xanthene dehydrogenase) and 48 human phase II metabolizing enzymes (acetyltransferase, acyl-CoA:amino acid N-acyltransferase, UDP-glucuronosyltransferase, glutathione S-transferase, methyltransferase, and sulfotransferase) were prepared. The mRNA expression level of each target enzyme was analyzed in total RNA from single and pooled specimens of various human tissues (adrenal gland, bone marrow, brain, colon, heart, kidney, liver, lung, pancreas, peripheral leukocytes, placenta, prostate, salivary gland, skeletal muscle, small intestine, spinal cord, spleen, stomach, testis, thymus, thyroid gland, trachea, and uterus) by real-time reverse transcription PCR using an ABI PRISM 7700 Sequence Detection System. Further, individual differences in the mRNA expression of representative human phase I and II metabolizing enzymes in the liver were also evaluated. The mRNA expression profiles of the above phase I and phase II metabolizing enzymes in 23 different human tissues were used to identify the tissues exhibiting high transcriptional activity for these enzymes. These results are expected to be valuable in establishing drug metabolism-mediated screening systems for new chemical entities in new drug development and in research concerning the clinical diagnosis of disease.