While primary human hepatocytes are considered the “gold standard” for the evaluation of human P450 induction, animal hepatocytes are useful for the evaluation of species-differences and for the selection of the animal species most relevant to human for in vivo studies. To emphasize the property of the animal species and to minimize individual differences, we developed a procedure for the preparation of cryopreserved monkey hepatocytes pooled from multiple donors. The monkey hepatocytes were isolated from multiple donors (N=40; 20 males and 20 females) on the same day. The hepatocytes were pooled followed by cryopreservation. The resulting “multiple donor pooled” monkey hepatocytes were found to have high viability. Most importantly, the hepatocytes retained their ability to be cultured as confluent, monolayer cultures (“Plateable”). We report here the application of the multidonor pooled cryopreserved Cynomolgus monkey hepatocytes in the evaluation of P450 induction. This study was conducted to compare monkey and human primary hepatocytes to response to prototypical drug inducers such as rifampicin (Rif), omeprazole (Ome), Phenobarbital (PB) and 3-Methylcholanthrene (3-MC) on the CYP1A and CYP3A gene expression. Pooled primary monkey hepatocytes (20 male and 20 female) or pooled primary human hepatocytes (5 male and 5 female) were treated with Rif (0.16, 1.25 and 10 uM), Ome (0.7, 6.25, 50 uM), PB (31.5, 125, 250 uM), or 3-MC (0.025, 0.25, 2 uM) for two days following by q-PCR quantification of mRNA of CYP1A1 and CYP3A8 for monkey, and CYP1A2 and CYP3A4 for human. The cryopreserved monkey hepatocytes or cryopreserved human hepatocytes were plated into a 24 well plate at a density 350,000 cells/well and allowed to attach for 4 hrs. Following attachment plating medium was removed and the cells were overlaid with 0.25 mg/ml Matrigel™ in plating medium and incubated for an addition 20 hrs. The plate was cultured in an incubator maintained with a humidified atmosphere of 95% air and 5% carbon dioxide. The various doses of Rif, PB, Ome or 3-MC were dissolved in 0.1% DMSO. The cells without administration of drugs and only with 0.1% DMSO was used as control. Total RNA was isolated individually from each well followed by cDNA synthesis. Real-time reactions were carried out using Taqman primers. Each PCR cycle threshold (Ct) was normalized to the average Ct of the endogenous housekeeping control gene GAPDH. The comparative ? Ct method was used to calculate relative quantification of gene expression. Treatment of Ome and 3-MC caused dose dependent induction of CYP1A1 in monkey and CYP1A2 in human hepatocytes. Induction fold of CYP1A2 in human hepatocytes was found to be higher than that for CYP1A1 in monkey hepatocytes. Treatment with Rif and PB caused dose dependent induction of CYP3A8 in monkey and CYP3A4 in monkey hepatocytes. The results suggest that monkey hepatocytes responded similarly to human hepatocytes to P450 inducers. The multiple donor pooled monkey hepatocytes should be useful for a case-by-case evaluation of the validity of the Cynomolgus monkey as an in vivo model for the prediction of human clinical findings.