A molecular method based on the detection of mitochondrial DNA from various animal species was developed to track the origin of surface water pollutions, and to differentiate human and animal sources. Mitochondrial DNA sequences were used to design PCR primers specific for human, bovine, ovine and porcine DNA using single, multiplex and nested PCR protocols. The primers were tested with DNA extracted from untreated domestic sewage, agricultural soils run-off, swine farm effluents and water from two rivers with known pollution sources. At least one of the four species was detected in most of these samples. The limit of detection in wastewater was 10(3)-10(4) cells L(-1) with a multiplex PCR protocol. This is the first report of a method using eukaryotic genetic DNA to detect and differentiate animal DNA from fecal sources in water. This innovative method is simple and could be used to quickly differentiate sources of pollution in a watershed.