Background: Some antigens of H. pylori are excreted into the stool of infected people. These antigens can be used to detect the infection by immunoassays such as ELISA. Our aim was to identify these antigens by immunoblotting and affinity chromatography techniques. Methods: Four different antigenic preparations, namely, whole cell sonicate (WCS), outer membrane proteins (OMPs), cytoplasmic antigens (CAs) and cell surface-associated antigens (CSAAs) were obtained from H. pylori. Rabbit antiserum against these preparations was used to detect them in fecal antigenic extracts (FAEs) of infected patients. Results: By immunoblotting, we were able to detect a 26 kDa band in the positive stool samples. Anti-OMPs acted more specifically, so, it was used to isolate the Helicobacter pylori diagnostic antigens (HpDAs) from the stool. More antigens (at least 4 antigens with the molecular weights of about 14, 26, 55 and 57 kDa) were isolated by affinity chromatography. But, the 26 kDa antigen had a higher concentration and was seen in almost all positive samples. Conclusion: Since the 26 kDa antigen is detectable by these two techniques in all positive samples, we are confident that this antigen is one of the major antigens of H. pylori, which is released into the stool and can be considered as a candidate diagnostic antigen to be used in diagnostic kit development.