Aims: To detect both free and plankton-associated Helicobacter pylori in seawater samples collected on the Italian coast of the Adriatic Sea using a nested-PCR. Methods and Results: Dissolved oxygen, pH, salinity and chlorophyll 'a' were the parameters recorded together with the characterization of zooplanktonic organisms. Plankton-associated H. pylori DNA was searched for in water samples filtered through 200 and 64 mum nylon nets whereas free bacteria were retained with the subsequent filtration through 0.22 mum pore-size membranes. Nested-PCR using primers for the glmM (ureC) gene was performed to reveal the presence of H. pylori. The DNA sequencing of amplified products confirmed the specificity of the assay. The sensitivity of the nested-PCR assay for H. pylori detection was 62 CFU per 100 ml in spiked water samples. Helicobacter pylori either free or bound to planktonic organisms was found in seven of 12 monthly samples. In particular, free bacteria were detected during the summer sampling and in November, December and March associated to planktonic cells. Conclusions: The presence of free and plankton-associated H. pylori in seawater suggests that it can be a significant reservoir and a potential route of transmission for the microorganism. Significance and Impact of the Study: Our study seems to provide a promising background to define new and effective strategies for surveillance of this human pathogen.