International and U.S. guidance for developmental neurotoxicity (DNT) testing of chemicals are provided by OECD DNT test guideline 426, OECD extended one-generation reproductive toxicity study (EOGRTS) test guideline 443, and U.S. EPA DNT test guideline 870.6300. These guidelines with their multiple behavioral and neuropathology endpoints are among the most logistically complex, labor-intensive bioassays conducted for regulatory purposes. This article identifies key issues in the practical implementation and analysis of neurobehavioral and neuropathology endpoints, with emphasis on quantitative brain morphometry testing requirements. This article also reviews the development of DNT guidelines, strengths and limitations of validation efforts, regulatory triggers for DNT testing, and recent guidance from government and workgroup efforts to enhance conduct and interpretation of studies for regulatory risk assessment purposes.