Current cancer risk assessment guidance recommends that mode-of-action (MOA) data be used to inform the qualitative and quantitative assessment of human health risk. However, the ideal MOA data rarely exist, and as a result there are few examples for which MOA information has been used quantitatively in risk assessment. A comprehensive MOA research program was recently undertaken for hexavalent chromium [Cr(VI)] to better understand the key events underlying the tumorigenic response observed in rodents exposed chronically to Cr(VI) in drinking water. In 2009, and at the direction of an independent science advisory board, MOA Framework guidance was used to identify data gaps and develop a research program that included a pharmacokinetic (PK) study to generate the data necessary to model tissue dose, dose-response and quantify interspecies variability, and a subchronic study in both rats and mice (including an interim 7 day time point), with evaluation of the toxicogenomics in target tissues, histopathology, biochemical measures of immune response and oxidative stress, analyses of DNA damage (8- OH-dG and Cr-DNA adducts), and in vivo mutation. The findings of these studies are used to identify and temporally sequence key events in the MOA and provide information for extrapolation across species and doses. The use of these MOA and PK studies for Cr(VI) cancer risk assessment are discussed as an example case study for using MOA data to refine human health risk assessment.