Mutagenic by-products produced by chlorination of humic substances have been examined. Soil humic substances were obtained from a commercial source and also extracted from a local soil. Ether extracts recovered from chlorinated humic substances were subjected to thin layer chromatography (TLC) fractionation followed by high-performance liquid chromatography (HPLC) analysis. Results from TLC fractionation showed that chlorination of humic substances yielded polar and relatively non-polar mutagenic by-products. Polar mutagenic by-product(s) accounted for between 65 and 76% of the total mutagenic activity of the fractions separated by TLC, whereas the activity of relatively non-polar products accounted for only 14–23%. HPLC analysis shows that the chlorinated humic extracts give one and two mutagenic peaks for the polar and relatively non-polar TLC fractions, respectively. No qualitative difference in HPLC pattern of mutagenicity was seen among the humic substances used.