BACKGROUND: Mucosal bleeding is frequently used as a measure of gastric mucosal integrity. We compared the orthotolidine method for quantifying haemoglobin in gastric juice with a method based on measurement of fluorescent porphyrins extracted from haem. We also investigated whether acid or pepsin had a deleterious effect on the results of either method. METHODS: We compared the effects of pH (2 or 7), time (up to 180 min), haemoglobin concentration and the addition of pepsin (2.5, 5 or 7.5 kU/mL hog pepsin) on the accuracy of the two methods. RESULTS: With the orthotolidine method there was a time-dependent decline in detectable haemoglobin concentration (P < 0.02) at pH 2 that was not seen at pH 7. The time- and pH-dependent decline in detectable haemoglobin was not seen using the porphyrin assay. CONCLUSION: The widely used orthotolidine method for determination of blood in gastric juice is less reliable than the porphyrin method as it is more likely to be influenced by acidic conditions in the stomach.