As demonstrated in Part I, cultured MutaMouse primary hepatocytes (PHs) are suitable cells for use in anin vitrogene mutation assay due to their metabolic competence, their “normal” phenotype, and the presence of the MutaMouse transgene for reliable mutation scoring. The performance of these cells in anin vitrogene mutation assay is evaluated in this study, Part II. A panel of 13 mutagenic and nonmutagenic compounds was selected to investigate the performance of the MutaMouse PHin vitrogene mutation assay. The nine mutagens represent a range of classes of chemicals and include mutagens that are both direct‐acting and requiring metabolic activation. All the mutagens tested, except for ICR 191, elicited significant, concentration‐dependent increases in mutant frequency (MF) ranging from 2.6‐ to 14.4‐fold over the control. None of the four nonmutagens, including two misleading, or “false,” positives (i.e., tertiary butylhydroquinone [TBHQ] and eugenol), yielded any significant increases in MF. The benchmark dose covariate approach facilitated ranking of the positive chemicals from most (i.e., 3‐nitrobenzanthrone [3‐NBA], benzo[a]pyrene [BaP], and aflatoxin B1[AFB1]) to least (i.e.,N‐ethyl‐N‐nitrosourea [ENU]) potent. Overall, the results of this preliminary validation study suggest that this assay may serve as a complimentary tool alongside the standard genotoxicity test battery. This study, alongside Part I, illustrates the promise of MutaMouse PHs for use in anin vitrogene mutation assay, particularly for chemicals requiring metabolic activation. Environ. Mol. Mutagen. 60:348–360, 2019. © 2019 The Authors.Environmental and Molecular Mutagenesispublished by Wiley Periodicals, Inc. on behalf of Environmental Mutagen Society.