Whole-genome transcriptome measurements are pivotal for characterizing carcinogenic mechanisms of chemicals and predicting toxic classes, such as genotoxicity, from in vitro and in vivo assays. DNA microarrays have evolved as the gold standard for this purpose. In recent years deep sequencing technologies have been developed that hold the promise of measuring the transcriptome with RNA-seq in a more accurate and unbiased manner than microarrays. So far, however, few applications have been published that assess the performance of RNA-seq within a toxicogenomics context. Here, we applied RNA-seq for the characterization of the in vitro transcriptomic responses in HepG2 cells upon exposure to benzo[a]pyrene (BaP), a well-known DNA damaging carcinogen. We demonstrate the performance of RNA-seq with respect to the identification of differentially expressed genes and associated pathways, in comparison with microarray technology. RNA-seq data generates more complete and thus accurate data on differentially expressed genes and affected pathways than microarrays. Additionally, we highlight the potential of RNA-seq for characterizing mechanisms related to alternative splicing and thereby gathering new information. Exposure to BaP alters the isoform distribution for many genes, including regulators of cell death and DNA repair such as TP53, BCL2 and XPA, which are relevant for genotoxic responses. Finally, we demonstrate that RNA-seq enables to investigate allele-specific gene expression, although no changes for that could be observed. Our results provide evidence that RNA-seq is a powerful tool for toxicology which, compared to microarrays, is capable of adding valuable information at the transcriptome level for characterizing toxic effects caused by chemicals.