AbstractWe have developed an in vitro mutation assay using primary hepatocytes from the transgenic Muta™Mouse. Primary hepatocytes were isolated using a two‐step perfusion method with purification by Percoll, cultured, and treated with benzo[a]pyrene (BaP), 2‐amino‐1‐methyl‐6‐phenyl‐ imidazo[4,5‐b]pyridine (PhIP), 3‐nitrobenzoanthrone (3‐NBA), and cigarette smoke condensate (CSC). The mean lacZ mutant frequency (MF) for the solvent control was approximately twofold greater than the spontaneous MF observed in liver tissue. A concentration‐dependent increase in MF (up to 3.7‐fold above control) was observed following exposure to BaP. Fourfold and twofold increases in mutant frequency were observed for 3‐NBA and PhIP exposures, respectively, without the addition of any exogenous metabolic activation. A slight but statistically significant increase in lacZ MF was observed for CSC, but only at the lowest concentration. This is the first report demonstrating that mutations can be detected in cultured primary hepatocytes from Muta™Mouse. The preliminary results presented suggest that the Muta™Mouse primary hepatocyte mutagenicity assay can be used as a cost‐effective tool for screening of environmental mutagens and therapeutic products. Environ. Mol. Mutagen., 2010 © 2009 Wiley‐Liss, Inc.