AIM: To compare the performance of three commercially available anti-human epidermal growth factor receptor 2 (HER2) antibodies in whole-tissue sections and tissue microarrays (TMAs) of a series of gastric tumors. METHODS: We present a comparative analysis of three anti-HER2 antibodies (HercepTest, 4B5 and SP3) using TMA and whole-tissue sections prepared from the same paraffin blocks of 199 gastric adenocarcinomas operated upon between January 2004 and December 2008 at a Brazilian cancer hospital. The data on the patients' age, sex, the anatomical location of the tumor and the Lauren's histological classification were collected from clinical and pathological records. The immunohistochemical (IHC) results were examined by two pathologists and the cases were classified as positive (3+), equivocal (2+) and negative (0 or 1+), according to the criteria of the IHC scoring system of gastric cancer. TMAs and whole-tissue sections were evaluated separately and independently. All cases yielding discordant IHC results and/or scored as 2+ were subjected to dual-color in situ hybridization in order to determine the final HER2 status. Besides determining the sensitivity and predictive value for HER2-positive status, we measured the accuracy of each antibody by calculating the area under the receiver operating characteristic (ROC) curve. The agreement between the results obtained using the TMAs and those obtained using the whole-tissue sections was assessed by means of Kappa coefficient. RESULTS: Intratumoral heterogeneity of HER2 expression was observed with all antibodies. HER2-positive expression (3+) in the whole-tissue sections was observed in 23 cases (11.6%) using the 4B5 antibody, in 18 cases (9.1%) using the SP3 antibody and in 10 cases (5.1%) using the HercepTest antibody. In the TMAs, 11 positive cases (5.6%) were identified using SP3 antibody, 9 (4.6%) using the 4B5 antibody and 6 (3%) using the HercepTest antibody. The sensitivity using whole-tissue sections and TMA, respectively, was 95.2% and 42.9% with 4B5, 90.5% and 66.7% with SP3 and 47.6% and 42.9% with HercepTest. The accuracy, calculated from the area under the ROC curve, using whole-tissue sections and TMA, respectively, was 0.91 and 0.79 by 4B5, 0.86 and 0.80 by SP3 and 0.73 and 0.71 by HercepTest. The concordance of the results obtained using whole-tissue sections and TMA was 97.4% (Kappa 0.75) using HercepTest, 85.6% (Kappa 0.56) using SP3 and 84.1% (Kappa 0.38) using 4B5. CONCLUSION: The use of the 4B5 antibody on whole-tissue sections was the most accurate IHC method for evaluating HER2 expression in gastric adenocarcinoma.