The lethal action and the effect from the removal of molecular sulfur by copper treatment were investigated in the Salmonella mutagenicity test for river sediments. Survival ratios of Ames Salmonella strains for five different sediment extracts were tested, and it was found that many of the river sediments showed the lethal action on TA98 and TA100, and the lethal action was stronger in the sample from which larger amount of extracts per unit weight were obtained. It was also found that the lethal action was reduced with the addition of the metabolizer S9 mix, and TA98 was more resistant than TA100 against the lethal substances in sediment extracts. Further, removal of molecular sulfur, one of the lethal substances, by copper treatment raised survival ratios of the tester strains. On high-pressure liquid chromatographic fractionation, the lethal substances including molecular sulfur distributed in non-polar and slightly polar fractions, and medium or high polar fractions did not reveal lethal action. Consequently, the removal procedure of molecular sulfur was proper for applying on the fraction contained non-polar and slightly polar compounds.