Collaboration was established in 2001 to evaluate a commercially available immunohistochemistry assay kit for the detection of bovine spongiform encephalopathy (BSE) disease-associated prion protein in formic acid-treated formalin-fixed samples of bovine brain. The kit protocol was evaluated at the National Centre for Foreign Animal Diseases (Winnipeg, Canada) and the Veterinary Laboratories Agency (Weybridge, U.K.). The U.K. laboratory provided paraffin-embedded blocks of brainstem (medulla oblongata at the level of the obex) from 100 positive cases defined by clinical signs and histopathology, and 100 clinically suspect but BSE-negative samples defined by histopathology and immunohistochemistry with anti-PrP monoclonal antibody R145. The Canadian laboratory provided 400 blocks from surveillance cases defined as clinically suspect but negative by histopathology and immunohistochemistry with anti-PrP antibody 6H4. Consecutive sections from each block were cut and coded. Each set of 600 slides was immunolabeled and read in each laboratory. Evaluation parameters included estimates of diagnostic sensitivity and specificity and reproducibility of the results. The kit performed with 100% sensitivity, specificity, and reproducibility in spite of minor differences between the laboratories in brain sample areas, fixation and processing, and in the immunolabeling protocol. Although enzyme linked immunosorbent assays are widely used in high throughput surveillance programs, standardized protocols and reagents for manual immunohistochemistry provide a useful adjunct to surveillance efforts, particularly in laboratories testing small numbers of samples or using immunohistochemistry for confirmation and characterization of BSE cases.