The presence of Helicobacter pylori (H. pylori) in the natural environment has been demonstrated in a number of studies. However, its route of infection into humans is unknown. To study this further, we attempted to detect H. pylori in the natural environment in a region of Japan with a high infection rate. Tap and well water and field soil samples were collected from around the residences of subjects who had participated in an epidemiological survey in 1996. Samples of water from rivers and ponds, and specimens of flies and cow faeces were collected in the region. DNA was extracted from the water, field soil and faecal samples after selective collection of H. pylori by the immunomagnetic-bead separation technique. H. pylori-specific DNA was detected in water, field soil, flies and cow faeces by nested polymerase chain reaction (PCR), and the ureA partial sequences of the PCR products were aligned. The nucleotide sequences of the samples amplified by PCR were highly homologous (96-100%) with the H. pylori sequence in the GenBank database and the H. pylori-specific DNA sequences were highly homologous with each other. These findings suggest the existence of H. pylori in the natural environment and a possible transmission route.