Humic substances (HS) are ubiquitous in the environment. However, some studies indicate that HS can induce a direct adverse effect on human health and hormone-like effects on fish, amphibians and invertebrates. In the present study we investigated possible biochemical mechanism of HS toxicity using reporter gene in vitro bioassay with H4IIE.luc cells. H4IIE.luc are stably transfected hepatoma cells with a luciferase reporter gene that is activated via cytosolic aryl hydrocarbon receptor (AhR). The most potent activator of AhR is 2,3,7,8-tetrachlorodibenzo-p-dioxin, but number of structurally diverse compounds has also been found to activate AhR. Aim of this study was to evaluate the dioxin-like activity of humic substances standards purchased from IHSS and Sigma-Aldrich companies. HS were dissolved in 0.05M NaOH, subsequently, lipophilic compounds (including fragments of HS) were extracted by liquid-liquid extraction with hexane/dichloromethane. Organic extract of HS was further treated with sulphuric acid to study the role of possible trace persistent contaminants (e.g. PCDDs, PCBs...). Dioxin-like activities of alkali solutions, organic extract and acid treated extracts have been evaluated using H4IIE.luc cells. Levels of possible known AhR-active contaminants were determined by GC-MS. Our results showed that both aqueous solutions and organic extracts of five out of twelve tested HS were active in luciferase assay. These results clearly indicated that some HS (or their fragments) might elicit significant inductions of AhR mediated effects. This might possibly lead to consequent health hazards.