Relatively little progress has been made in understanding the nature of the Reed-Sternberg (RS) cell and its morphologic variants in Hodgkin disease (HD). This is primarily due to the fact that RS cells represent a minute subpopulation within HD lesions. To investigate the clonal origin of RS cells and variants, we studied 27 HD lesions obtained from 11 patients. Using an image analyzer (CAS 200) we were able to demonstrate that CD30-positive RS cells are clonal elements with unique and individualized DNA profiles and that the DNA content of any given patient RS cell population is constant over time and in different pathologic sites. Using 1, 9, 11, and X alpha satellite chromosome probes and interphase cytogenetics, we also demonstrated that RS cells obtained from different tissue samples of the same patient have a unique and often abnormal chromosomal pattern. To definitively prove the hypothesis that CD30-positive RS cells are clonal elements, we investigated the presence of point mutations within p53 gene exons 5 through 9 and found that only a single patient possessed a nonsense p53 somatic point mutation (Arg to His). This same mutation could be identified in all of his available biopsies. Altogether, these findings demonstrate that RS cells and variants in HD are clonal and represent the neoplastic elements of this entity.