HER2/neu status in breast cancer is determined by immunohistochemical analysis and/or fluorescence in situ hybridization (FISH). Previous studies have found widely varying sensitivities and specificities for anti-HER2/neu antibodies, including recently developed rabbit monoclonal antibodies. The current prospective study compared rabbit monoclonal antibody SP3 and rabbit polyclonal antibody A0485 immunostaining on routinely processed consecutive cases of breast carcinoma. Of 1,610 cases tested, 261 (16.2%) equivocal (2+) cases were evaluated by FISH. Of 253 cases equivocal with A0485 results, 125 (49.4%) were negative with SP3. In 22 (8.7%) of 253 cases equivocal with A0485, there was amplification by FISH, and 3 of these cases were SP3- (0/1+). Of the 20 (14.8%) of 135 SP3-equivocal cases amplified by FISH, 1 case was A0485-. The reported false-negative rate with A0485 is 2.8%, and the American Society of Clinical Oncology/College of American Pathologists guidelines recommend a rate of less than 5%. Compared with A0485, the false-negative rate with SP3 is only 0.3% (3/1,156) higher, but it shows about a 50% reduction in equivocal scores, reducing the need for reflex FISH testing.