In order to characterize the expression of the viral interleukin-6 (vIL-6) homologue in various human herpesvirus 8 (HHV-8)-associated diseases, in situ hybridization and immunohistochemistry were applied to formalin-fixed specimens. These assays showed consistent expression of vIL-6 in primary effusion lymphomas and in a case of human immunodeficiency virus (HIV)-associated lymphadenopathy with a Castleman's disease-like appearance. In contrast, Kaposi's sarcoma specimens showed marked differences among specimens. In a consecutive series of specimens from the Johns Hopkins archives, vIL-6 expression was demonstrated in one of 13 cases. However, among 7 specimens selected from the AIDS Malignancy Bank because of their high levels of the T1.1 lytic transcript and virion production, vIL-6 expression was consistently demonstrated in infiltrating mononuclear cells and occasional spindle-shaped cells. Thus vIL-6 expression in clinical specimens correlates with other measures of the lytic viral cycle. Both assays generally give congruent results and are consistent with the possibility that vIL-6 expression plays a role in the pathogenesis of a variety of HHV-8-associated diseases.