Double immunodiffusion (Ouchterlony) tests gave regularly positive reactions when extracts of [35S]-methionine-labeled, n-butyrate-induced P3HR-1 cells were allowed to react with serum pools with high Epstein-Barr virus (EBV) antibody titers from Burkitt lymphoma or nasopharyngeal carcinoma patients, but not with EBV antibody-negative sera. Similarly prepared extracts of noninduced P3HR-1 cells gave no precipitation lines with the antibody-positive sera. The composition of the precipitates was analyzed by SDS-PAGE and compared with immunoprecipitates obtained by indirect immunoprecipitation in solution. Three lines precipitated on the Ouchterlony plate were analyzed; these lines were located at close (a), intermediate (b), and distant (c) positions in relation to the antigen well. Precipitate (a) contained polypeptides with molecular weights of 165,000 (165K), 152K, 138K, 134K, 103K, and 55K. Precipitate (b) contained 152K and 138K as the major components, while 55K was relatively underrepresented. Precipitate (c) contained 90K and 55K as major components, while 152K was a minor component. The method is suitable for the study of possible subtype differences between defined antigenic components of the early and late EBV-determined antigen complexes.