Risk assessment of chemical substances requires knowledge of the intrinsic toxicity (hazard), including their dose-response relationship and, if possible, their mechanisms of toxicity, and assessment of exposures to chemicals ie., route of exposure, sources, levels and temporal pattern. A wide range of exposure scenarios will be addressed, both involuntary (environmental and occupational) and voluntary (drug abuse), which determine the level of such exposure, from relatively low doses representing environmental pollutants in general population to highest exposures in workers and drug-abuse cases. Through different types of chemical substances, both classical and emerging persistent organic pollutants, pesticides, organic solvents, drugs of abuse, ... etcetera the molecular basis of the toxic effect and its impact on behavior, respiratory function or nervous system will be discussed among others. The relatively recent development of experimental models, which allows the reduction the use and sometimes replacement of experimental animals, has found its application in toxicological risk assessment for which representative examples of the application of such models will be presented. Beyond toxic effects, chemicals can be instrumentally used for identification of molecular targets and biomarkers of potential clinical and / or environmental applications.