The use of urease in enzyme-linked immunosorbent assays (ELISAs) offers the advantages of convenience and safety. However, urease-based ELISA, performed in standard microtitre format, could result in false positive reactions upon prolonged incubation. False positive reactions appeared when wells containing substrate solution absorbed ammonia liberated from a reactive well nearby. Thus, the intensity of the false reaction was proportional to that of the urease reaction. The transfer of ammonia was demonstrated by pyrolysis-mass spectrometry. When urease conjugates were compared with peroxidase conjugates in the detection of IgG and IgM, there was no evidence that one enzyme was superior to the other in terms of increasing the sensitivity or the speed of ELISA.