BACKGROUND: Different culture mediums were used for the isolation of Helicobacter pylori. In this study three mediums commonly used for the culture of this microorganism were evaluated: chocolate agar, Martin-Lewis medium and CP of Dent medium as well as a new selective medium Pylori agar. METHODS AND RESULTS: Over a period of 7 months gastric biopsies were collected in 112 patients with clinical manifestations of chronic atrophic gastritis. H. pylori was isolated in some of these mediums on 73 occasions (65.1%). On 72 of the 73 occasions the microorganism grew in the Pylori agar medium (98.6%) being the medium achieving the greatest number of isolations, followed by the CP of Dent medium with 67 isolations (91.7%), 65 isolations with the Martin-Lewis medium (89.0%) and 57 isolations with chocolate agar (78.0%). The chocolate agar medium was that which most frequently presented contaminant flora (35 times: 61.4%) and the CP of Dent medium achieved the most rapid and widespread growth. The colonies were largest and therefore easier to recognize in the CP of Dent and Pylori agar mediums. CONCLUSIONS: The authors recommend the association of the CP of Dent and Pylori agar mediums in the isolation of Helicobacter pylori since these two mediums provide the greatest number of isolations with the greatest growth and ease for colony recognition.