Background. The rapid urease test (RUT) is used at Groote Schuur Hospital for diagnosing Helicobacter pylori infection. This is an in-house method, which has not been validated. Objective. To validate our practice of reading the RUT immediately after endoscopy (RU[T.sub.0]), by comparing this with a reading at 24 hours (RU[T.sub.24]) and with histological analysis. Design. Ninety consecutive patients undergoing upper endoscopy over a 6–week period from October 2005 to November 2005, and in whom rapid urease testing was indicated, were included in the study. Patients with recent exposure (within 2 weeks of endoscopy) to proton pump inhibitors (PPIs), histamine receptor antagonists ([H.sub.2]RAs) and antibiotics (confounders) were noted and included in the cohort. Two antral and two body biopsies were taken for histological examination and a third antral biopsy was placed in the RUT bottle. Both haematoxylin and eosin and modified Giemsa staining methods were used to identify H. pylori. The RUT was read immediately (within 5 minutes of upper endoscopy) (RU[T.sub.0]), as per our current practice, and each specimen was re-read at 24 hours (RU[T.sub.24]). Sensitivity, specificity, positive and negative predictive values and the impact of confounders were calculated. Results. Of the 90 patients undergoing rapid urease testing, 39% were male and 61% were female, with a mean age of 55 years (range 22-79 years). Histological examination revealed H. pylori in 67.8% (N=61) of the biopsy specimens. In the 65 patients without confounders, the sensitivity and specificity of the RU[T.sub.0] were 65.9% and 100% respectively, and 90.9% and 100% for RU[T.sub.24]. After including the 25 patients with confounders, the sensitivity and specificity were 68.8% and 100% for RU[T.sub.0], and 90.1% and 100% for RU[T.sub.24] respectively. Thirteen RU[T.sub.0] specimens (30.9%) that were initially negative became positive at the RU[T.sub.24] reading. There were 6 (9.8%) RU[T.sub.0]- and RU[T.sub.24]-negative but histology-positive specimens. Four of these 6 false-negative RU[T.sub.24] results could be accounted for by a low H. pylori density on histological analysis (2 patients were taking PPIs). Confounders did not alter the sensitivity and specificity outcomes or impact on the number of false-negative RUTs.