Human biomonitoring (HBM), defined as the measurement of concentrations of chemicals or of their metabolites in human biological matrices, is considered the method of choice for determining internal exposures in individuals. HBM is part of environmental exposure surveillance systems in several countries. In 2007, the Ministry of Agriculture, Food and Environment promoted BIOAMBIENT.ES project, a national-level HBM study on environmental pollutants carried out in Spain to estimate levels of heavy metals, persistent organic pollutants, and other substances on the Spanish active workforce. BIOAMBIENT.ES is a nationwide cross-sectional study, with a stratified cluster sampling designed to cover all geographical areas, sex and occupational sectors, and aimed to obtain a representative sample of the Spanish workforce. Participants were recruited among people residing in Spain for 5years or more, which underwent their annual occupational medical check-up in the health facilities of the Societies for Prevention of IBERMUTUAMUR, MUTUALIA, MC-PREVENCIÓN, MUGATRA, UNIMAT PREVENCIÓN, and PREVIMAC (March 2009-July 2010). A total of 1,892 subjects fulfilled the criteria for inclusion, donated biological samples (1,880 blood, 1,770 urine, and 577 hair) and completed a short self-administrated epidemiological questionnaire on environmental and lifestyle-related exposures. Additionally, clinical information from participant's health exams was obtained. This project will provide a first overview of the body burden of selected pollutants in a representative sample of the Spanish-occupied population. This information will be useful to establish reference values of the studied population and, eventually, to evaluate temporal trends and the effectiveness of environmental and health policies.