Abstract In ongoing work by this Project, we have found that developmental exposures to mitochondrial toxicants cause neurotoxic outcomes in Caenorhabditis elegans, including morphological alterations in neurons, altered behavior, and, in the long term, increased susceptibility to neurodegeneration. In line with the EPA, we describe all of these as “developmental neurotoxicity (DNT),” because they result from exposures that occur during development. Two important and overarching mechanisms of DNT are 1) changes to neurogenesis resulting in altered cell fate, morphology, and connectivity (“hardwiring”), and 2) persistent changes to the function of neurons that appear to be morphologically normal (epigenetic “programming”). Distinguishing these is challenging; we propose a novel and powerful way to assess each possibility. We will begin with an in vivo yet relatively high-throughput and economic model, C. elegans. C. elegans offers an additional, key benefit: developmental neurogenesis is normally invariant, permitting clear identification of variation in hardwiring as well as behavioral and stress-responsive changes without morphological alteration (programming). Work in C. elegans will be followed by testing in human neuronal stem cells (hNSCs) that permit human-relevant DNT testing, plus the opportunity to identify sex-specific differences and epigenetic modifications. Relatively few chemicals have been rigorously evaluated for DNT. The paucity of information is even more pronounced for chemical co-exposures, despite the fact that combined exposures are the reality. This lack of testing of mixtures results partly from regulatory policy, and partly from technical challenges in laboratory testing of co-exposures. Our combined in vivo-in vitro approach will permit us to rigorously test for DNT resulting from both complex environmental mixtures, and from defined combinations of individual Superfund chemicals that we will evaluate for non-additive effects. We will test the effects of the prototypical developmental neurotoxicants Pb, Cd, and polycyclic aromatic hydrocarbons, singly and in combinations dictated by known environmental concentrations. We will compare our outcomes in C. elegans and hNSCs, to those obtained by other Projects in fish, rats, and people. Demonstration that C. elegans can be reliably used to investigate mixture DNT will add a powerful new model for testing and regulation of environmental mixtures. Finally, we will test the degree to which mitochondrial dysfunction, key to neurodevelopment, drives DNT by these prototypical chemicals. These chemicals have multiple molecular targets, including but not limited to different mitochondrial macromolecules. The fact that these chemicals individually all affect mitochondria and neurons, but by different mechanisms, is why we predict synergistic interactions. However, while mitochondria are known targets of these chemicals, the extent to which mitochondrial toxicity drives their DNT is n...