Abstract Helicobacter pylori adherence to gastric tissue plays an important role in pathogenesis. Studies of H. pylori adherence mechanisms have utilized continuous cell lines grown in vitro. This creates a demand for a simple and reliable in vitro technique for measuring H. pylori adherence. We developed a new monoclonal antibody enzyme-linked immunosorbent assay (ELISA) technique and measured H. pylori adherence to three cell lines. The monoclonal antibody was specific for the H. pylori urease, an enzyme characteristic to H. pylori. The ELISA was an accurate, semiquantitative technique for measuring adherent H. pylori to continuous cell lines in culture. The ELISA was easy to perform and required 4 h from start to finish. ELISA data were compared with microscopic evaluation of parallel cultures grown in slide–well chambers and immunostained with biotinylated monoclonal antibody. The ELISA proved more sensitive than microscopic quantitation. H. pylori isolate-to-isolate differences in urease expression and its effect on the ELISA were investigated. The antibody reactivities with different isolates of H. pylori were evaluated by dot-blot immunoassay and gold stain for total protein. The results showed that the ELISA assay was prone to error due to differences in urease expression among H. pylori isolates. However, the dot-blot technique proved effective in predicting this error and can be used to adjust raw data for isolate-to-isolate comparisons.