Environmental chemicals have a potential impact on children's health as the developing brain is much more vulnerable to injury caused by different classes of chemicals than the adult brain. This vulnerability is partly due to the fact that very complex processes of cell development and maturation take place within a tightly controlled time frame. So different stages of brain development are susceptible to toxic effects at different time points. Additionally the adult brain is well protected against chemicals by the blood brain barrier (BBB) whereas the placenta only partially protects against harmful chemical exposure. Many metals easily cross the placenta and BBB barrier since even after the birth BBB is not entirely differentiated (until about 6 months after birth). Additionally, the susceptibility of infants and children is due to increased exposure, augmented absorption rates, and less efficient ability of defense mechanism in comparison to adults. The In Vitro Session during the 12th International Neurotoxicology Association meeting (Jerusalem, June, 2009) provided the opportunity to discuss the new challenges that have to be faced to create new type of safety assessments for regulatory requirements. The integration of various tests into testing strategies as well as combination of information-rich approaches with bioinformatics was discussed. Furthermore relevant models and endpoints for developmental neurotoxicity (DNT) evaluation using in vitro approach were presented. The primary neuronal cultures of cerebellar granule cells (CGCs) as well as 3D aggregate model and the possible application of human embryonic and adult stem cells was discussed pointing out the potential of these models to be used for DNT testing. The presented systems are relevant for DNT evaluation as the key processes of brain development such cell proliferation, migration and neuronal/glial differentiation are present. Furthermore, emerging technologies such as gene expression, electrical activity measurements and metabonomics have been identified as promising tools. In a combination with other assays the in vitro approach could be included into a DNT intelligent testing strategy to speed up the process of DNT evaluation mainly by initial prioritization of chemicals with DNT potential for further testing.