The mutagenicity of crude extracts and subfractions of two samples of a reference sewage sludge material and two sewage sludges from two wastewater treatment plants (WWTP), one urban and the other one urban mixed with industrial, was assessed using an Ames fluctuation assay based on 384-well microtiter plates with liquid cultures. Crude extracts of sludges were obtained by ultrasonic extraction with dichloromethane/methanol, and further column fractionation yielded two fractions, one of which containing mutagenic polycyclic aromatic hydrocarbons (PAHs). Quantitative analysis performed by gas chromatography-mass spectrometry gave sum concentrations of the 16 PAHs listed as priority pollutants by the U.S. Environmental Protection Agency at levels between 1,305 and 2,442 microg/kg. Subjecting crude extracts and column fractions to the mutagenicity assay with Salmonella strains TA98 and TA 100 provided good qualitative correlation between the presence of mutagenic PAH and the induction of gene mutations. In general, the crude extracts and the PAH-fractions induced positive responses in the assay with both bacterial strains on metabolic activation by S9 rat-liver homogenate, whereas direct-acting mutagens were not detectable. In the assay with the real sludge samples of two different WWTPs, TA98 proved to be more sensitive than TA100; however, similar sensitivities of the tester strains were observed for two reference sewage sludge materials of the same origin. The outcomes of the Ames fluctuation assay demonstrated its performance as a cost-effective and relatively rapid screening tool to assess the genotoxic potential of complex environmental samples.