The complexity of human biology has made prediction of health effects as a consequence of exposure to environmental chemicals especially challenging. Complex cell systems, such as the Biologically Multiplexed Activity Profiling (BioMAP) primary, human, cell-based disease models, leverage cellular regulatory networks to detect and distinguish chemicals with a broad range of target mechanisms and biological processes relevant to human toxicity. Here the authors use the BioMAP human cell systems to characterize effects relevant to human tissue and inflammatory disease biology following exposure to the 320 environmental chemicals in the Environmental Protection Agency’s (EPA’s) ToxCast phase I library. The ToxCast chemicals were assayed at 4 concentrations in 8 BioMAP cell systems, with a total of 87 assay endpoints resulting in more than 100,000 data points. Within the context of the BioMAP database, ToxCast compounds could be classified based on their ability to cause overt cytotoxicity in primary human cell types or according to toxicity mechanism class derived from comparisons to activity profiles of BioMAP reference compounds. ToxCast chemicals with similarity to inducers of mitochondrial dysfunction, cAMP elevators, inhibitors of tubulin function, inducers of endoplasmic reticulum stress, or NFκB pathway inhibitors were identified based on this BioMAP analysis. This data set is being combined with additional ToxCast data sets for development of predictive toxicity models at the EPA.