The use of in vitro human liver cell models is an attractive approach in toxicogenomic studies designed to analyze gene expression changes induced by a toxic chemical. However, in such studies, reliability, reproducibility and interlaboratory concordance of microarrays, as well as the choice of the most suitable cell model, remain a matter of debate. This work was aimed at evaluating the robustness of microarray technologies and the suitability of the highly differentiated human HepaRG cell line in the investigation of gene expression changes induced by a toxic compound in human liver. The influence of various experimental conditions including cell cultures grown at different test sites, different generations of microarrays, RNA analysis platforms and softwares, was tested on gene expression profiles induced by a 20h treatment with an 8mM concentration of phenobarbital as the toxic compound. As many as 1099 genes (p-value<0.01 and 1.5-fold-change), representing 74% and 30% of the signature genes detected with Agilent 22 and 44K pangenomic microarrays, respectively, were shown to be modulated in common in six independently performed experiments. The most modulated genes included both those known to be regulated by phenobarbital, such as cytochromes P450 and membrane transporters, and those involved in oxidative stress, inflammation and apoptosis, typifying a toxic insult. These data provide strong support for the use of a toxicogenomic approach for the in vitro prediction of chemical toxicity, and for the choice of human HepaRG cells as a promising model system for human hepatotoxicity testing.