A wide variety of contaminants derived from diesel and gasoline engines, tire, asphalt, and natural organic compounds is found in road dust. Polycyclic aromatic compounds (PACs) are the important toxic targets among various contents in road dust and diesel exhaust particulates (DEPs), and endocrine-disrupting activity of PACs was suggested. In the present study, aryl hydrocarbon receptor (AhR) ligand activity was confirmed in the extract of both road dust and DEPs. In the separation of the extracts for both road dust and DEPs with reversed-phase HPLC, it was found that polar fractions contributed to significant AhR ligand activity in both a mouse hepatoma (H1L1) cell system and a yeast system. Furthermore, the contribution of these polar fractions was higher in DEPs than in road dust, probably because of the greater concentration of oxy-PAHs in DEPs than in road dust. The contribution of contaminants associated with the polar region to AhR ligand activity was also evident following the separation of road dust with normal-phase HPLC. Additionally, remarkable estrogen receptor (ER) ligand activity was detected in the highly polar region separated with normal-phase HPLC. It is suggested that many unknown AhR or ER ligand active compounds are contained in the polar region.