The river Lambro is the most polluted tributary of the river Po in North Italy and was chosen as a representative water course discharging industrialized areas. Water and sediment samples of the river Lambro were investigated regarding the presence of endocrine disrupting compounds. A combined procedure was used consisting of solid-phase extraction and HPLC based fractionation of samples, of screening for (anti)androgenic activity using the yeast androgen screen (YAS) and of chemical analysis using HPLC-MS/MS and GC-MS. Androgenic and antiandrogenic activities were found in specific fractions of the water and sediment while the total extracts showed antiandrogenic activity only. The chemical analysis of the fractions and total extracts with antiandrogenic activities revealed the presence of compounds with suspected antiandrogenic potency such as bisphenol A, iprodione, nonylphenol, p,p'-DDE and tert-octylphenol but other unknown compounds contributed mainly to the observed antiandrogenic activities. The antiandrogenic load of the river Lambro ranged between 1.34 and 17.1 microM flutamide-equivalents and may pose a risk to aquatic environments. Future screenings for EDC in the environment that have the potential to interfere with reproduction of aquatic organisms should be extended to different modes of actions including (anti)androgenic ones.