While it is generally accepted that neonates and adults differ in drug metabolizing enzyme activities, there is little information on the differences in specific drug metabolizing enzyme pathways. In our laboratory, hepatocytes were isolated and cryopreserved from three neonates with enencephaly who had full-term in utero development, borne alive, would only survive for minutes to hours after birth due to the lack of brain functions. The hepatocytes retained high viability upon thawing (>80% based on trypan blue exclusion). The hepatocytes were evaluated for drug metabolizing enzyme activities and compared to pooled cryopreserved adult human hepatocytes, using the following pathway selective substrates: phenacetin (CYP1A2), coumarin (CYP2A6), bupropion (CYP2B6), paclitaxel (CYP2C8), S-mephenytoin (CYP2C19), diclofenac (CYP2C9), CYP2D6 (dextromethorphan), chlorzoxazone (CYP2E1), midazolam (CYP3A4), testosterone (CYP3A4), astemizole (CYP2J2), benzydamine (FMO), kynuramine (MAO), hydroxycoumarin (UGT and SULT), 4-aminobenzoic acid (NAT1), and sulfamethazine (NAT2). The activities in adult (A) and neonatal (N) human hepatocytes were, in pmol/min/million hepatocytes: CYP1A2: 59(A); 011-0.16(N); CYP2A6: 102 (A), 0.13-0.17 (N); CYP2B6: 29 (A), 0.35-0.50 (N); CYP2C8: 141(A), 0.19-0.52 (N), CYP2C9: 98(A); 2.3-7.0(N); CYP2C19: 20(A); not detected(N);CYP2D6: 17(A); 0.2-2.7(N); CYP2E1: 47(A); 26-28(N); CYP3A4-midazolam: 16(A); 1.3-3.5(N); CYP3A4-testosterone: 204(A), 5.6-27(N); UGT: 537(A), not detected(N); SULT: 20(A), 0.15-0.23(N); FMO: 761(A), 4.2-8.5(N); MAO: 1893(A), 24-47(N); NAT1: 9.2(A), 7.2-9.9(N); NAT2: 42(A); 0.11-0.74(N). The results showed that in general, neonatal hepatocytes had substantially lower drug metabolizing enzyme activities than adult hepatocytes, especially for CYP2C19 and UGT which were not detected in neonatal hepatocytes. One surprising finding is that CYP2E1 and NAT1 activities were similar for both adult and neonatal hepatocytes. Our finding may be useful in the consideration of choice of mediation and dosing regimens for neonates.