The lineage and clonality of Hodgkin's disease (HD) were investigated by analyzing the organization of the immunoglobulin and T-cell receptor beta-chain (T beta) gene loci in 18 cases of HD, and for comparison, in a panel of 103 cases of B- and T-cell non-Hodgkin's lymphomas (NHLs) and lymphoid leukemias (LLs). Sizable clonal B- or T-cell populations, representing greater than or equal to 10% of the pathologic sample, were readily detectable by immunogenotypic analysis in all 103 NHLs and LLs but not in any of the 18 cases of HD. However, extremely minor clonal populations (less than or equal to 1%) were detectable in 3 of 18 cases of HD. We demonstrated that these minor clonal populations do not correspond to Reed-Sternberg (RS) cells since clonal immunoglobulin or T beta gene rearrangements are not detectable in cases of HD containing greater than 25% RS cells. The number of RS cells present in these samples appeared to correlate directly with the pattern of gene rearrangements characteristic of polyclonal T cells. These studies demonstrate that Southern blot hybridization analysis for clonal immunoglobulin and T beta gene rearrangements represents an accurate, objective tool in the differential diagnosis between HD and NHL; that HD is predominantly composed of polyclonal B and T cells; that minor clonal B- or T-cell populations unrelated to RS cells occasionally can be found in HD; and that RS cells do not represent clonal B- or T-cell expansions. Finally, our data preliminarily suggest that RS cells may represent polyclonal T-cell populations.