Several have been performed to evaluate the mutagenicity of soil samples in urban and industrial areas. The use of uncontaminated reference areas has been an obstacle to the study of environmental mutagenesis. The study aimed to indicate a methodology to define reference areas in studies of environmental contamination based on "Ambient Background Concentration" of metallic elements associated with the Salmonella/microsome assay. We looked at three potential reference areas, two of them close by the industrial sources of contamination (São Jerônimo reference, near the coal-fired power plant, and Triunfo reference, near the wood preservative plant), but not directly influenced by them and an area located inside a protected area (Itapuã reference). We also carried out chemical analyses of some metals to plot the metal profile of these potential reference areas and define basal levels of these metals in the soils. After examining the mutagenicity of the inorganic extracts using strains TA98, TA97a, and TA100, in the presence and absence of S9 mix, we indicated the São Jerônimo reference and the Itapuã reference as two sites that could be used in future studies of mutagenicity of soils in southern Brazil. The association between a mutagenicity bioassay and the "Ambient Background Concentration" seems to be a useful method to indicate the reference areas in studies of contamination by environmental mutagens, where these results were corroborated by canonical correspondence analysis.