Variation in assay sensitivity was studied in more than 90 laboratories that assayed 4formalin-fixed, paraffin-processed breast and ovarian carcinoma cell lines with graded levels of HER-2/neu protein overexpression and known levels of HER-2/neu gene amplification, in addition to breast carcinomas fixed and processed in the laboratories. Main methods were the HercepTest (DAKO, Ely, England) and individualized protocols using a polyclonal antibody and the CB11 clone. While the proportion of laboratories achieving appropriate results with the HercepTest was significantly higher than for participants using other assays, laboratories using other assays showed significant improvement in the second assessment run. The level of agreement in evaluations by 26 laboratories using the HercepTest was excellent on cell lines and tumors and was significantly greater than that achieved by the remaining 41 laboratories using other immunohistochemical methods. While laboratories using the DAKO HercepTest had the highest level of reproducibility in assay sensitivity and evaluation, the significant improvement in results by laboratories using other antibodies in the second assessment run suggests that stringent quality control and an ongoing quality assurance program using a standard reference material have the potential to improve the reliability of immunohistochemical assays for HER-2/neu, regardless of the antibody used.