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The developed method for mercury speciation analysis has been validated and used for the biomonitoring study of mercury species in human hair. Statistical evaluation proved the reliability of simplified determination of inorganic mercury (difference between total mercury and methylmercury). The results of the validation showed that the method is very well suitable for the determination of both species of mercury in hair for biomonitoring purposes. Non-exposed schoolchildren from three areas in the western and central part of the Czech Republic were chosen as the target group. Tenth of a microgram per gram of the total mercury were generally found in the analyzed hair; values higher than 1μg g−1 were detected only exceptionally. Comparable results were obtained for two western areas and differed significantly from those for the third area located in the central part of the Czech Republic. In the areas examined, the mean methylmercury contents amounted to 23–46% of the total mercury in the hair. The results confirm an assumption that exposure to mercury does not pose a significant risk to the population in the Czech Republic.