To better characterize the clonality and pathogenesis of Hodgkin's disease (HD), we used polymerase chain reaction (PCR) and Southern blot to analyze the rearrangement of immunoglobulin (Ig) and T-cell receptor (TCR) genes, the bcl-2 oncogene, and the Epstein-Barr virus (EBV) genotype. In situ hybridization studies of EBV were also done. Twenty-six cases of HD were compared with 15 cases of non-specific lymphadenitis, 7 with incipient adult T-cell leukemia/lymphoma (ATLL), and 4 T-cell rich B-cell lymphomas (TRBL), all of which histologically resembled HD. EBV genes were detected in 20 of 26 HD patients (77%) and in 7 of 15 patients with non-specific lymphadenitis (47%), 5 of 7 with incipient ATLL (71%), and 1 of 4 with TRBL (25%). In contrast to specimens of non-specific lymphadenitis, TRBL, and incipient ATLL, only one EBV genotype was evident in the specimens of HD. EBV latent membrane protein (LMP) was detected immunologically in 16 of 26 HD patients (62%), one of four TRBL (25%) and one of seven incipient ATLL (14%), but it was not evident in non-specific lymphadenitis. The LMP positive cases showed amplified EBV genomes. Only one of the 26 cases of HD had a bcl-2 gene rearrangement by PCR, but this was not seen in any other disease. The bcl-2 protein was detected immunologically in seven of the 26 HD patients (27%) and in one of the seven incipient ATLL cases (14%). EBV has been reported to upregulate bcl-2 expression, but in this study the presence of bcl-2 protein did not correlate with the presence of the t(14;18) translocation or EBV-LMP. All TRBLs showed rearrangement of the immunoglobulin genes by PCR and/or Southern blot, and the giant cells were of B-cell type. All incipient ATLLs displayed rearrangement of the TCR genes, and the giant cells were of T-cell origin. In seven of 26 HD cases, the giant cells were weakly stained with T-cell antibodies, in another seven positive with B-cell antibodies and in 18 instances polyclonally positive for both kappa and lambda. However, PCR and Southern blot displayed only two cases of TCR gene rearrangement, while two others had very weak rearrangements of immunoglobulin gene positive only by PCR. Thus the T and B-cell genotype did not correlate with the T and B-cell phenotype recorded in these cases. The absence of Ig and TCR gene rearrangements seems to be common in HD, compared with in TRBL and incipient ATLL.