Effect-directed analysis (EDA) is an approach used to identify (unknown) contaminants in complex samples which cause toxicity, using a combination of biology and chemistry. The goal of this work was to apply EDA to identify developmental toxicants in soil samples collected from a former municipal landfill site. Soil samples were extracted, fractionated, and tested for developmental effects with an embryotoxicity assay in the zebrafish Danio rerio. Gas chromatograph mass selective detection (GC-MSD) chemical screening was used to reveal candidate developmental toxicants in fractions showing effects. In a parallel study, liquid chromatography-hybrid linear ion trap Orbitrap mass spectrometry was also applied to one polar subfraction (Hoogenboom et al. J. Chromatogr. A2009, 1216, 510-519). EDA resulted in the identification of a number of previously unknown developmental toxicants, which were confirmed to be present in soil by GC-MS. These included 11H-benzo[b]fluorene, 9-methylacridine, 4-azapyrene, and 2-phenylquinoline, as well as one known developmental toxicant (retene). This work revealed the presence of novel contaminants in the environment that may affect vertebrate development, which are not subject to monitoring or regulation under current soil quality assessment guidelines. © 2011 American Chemical Society.