Although Epstein-Barr virus (EBV) infection has been implicated in the development of a subset of Hodgkin's disease, the etiology of EBV-negative Hodgkin's disease remains to be determined. We investigated the status of human cytomegalovirus (hCMV) infection in 42 cases of Hodgkin's disease in a Chinese population. Using a nested polymerase chain reaction assay, nine of the 42 cases (21.4%) were found to contain EBV DNA. While only one of the nine EBV-positive cases demonstrated amplifiable hCMV DNA, approximately 33.3% of the EBV-negative cases (11/33) showed molecular evidence of hCMV infection (8 mixed cellularity; 2 nodular sclerosis; and 1 lymphocyte depletion). All 17 lymph nodes with non-specific reactive changes were negative for hCMV DNA. The presence of hCMV in Reed-Sternberg cells and their variants was further confirmed by single-cell polymerase chain reaction, in situ hybridization and immunohistochemical analysis, which demonstrated ~50% amplified hCMV DNA. These results suggest for the first time that hCMV infection plays a potential role in signaling the cell transformation and pathogenesis of Hodgkin's disease, particularly in EBV-negative cases.