Post-transplantation lymphoproliferative disorder (PTLD) is a serious complication of organ transplantation. The majority of these lymphoid expansions are associated with latent Epstein-Barr virus (EBV) infection, but lytic activity may play an important role in initiating the disease process. Forty-eight specimens from 44 allograft recipients with EBV-associated PTLD were studied by immunohistochemical techniques for EBV lytic proteins, by use of monoclonal antibodies specific for the immediate early latent to lytic switch protein BZLF1 and two early antigens (diffuse and restricted). In addition, the specimens were studied by in situ hybridization for EBV DNA by use of an oligonucleotide probe for the EBV NotI tandem DNA repeats, whose expression seems confined to lytic infection. Thirty specimens were studied by in situ hybridization for immediate early (BZLF1) and late (gp350/220) lytic mRNA transcripts. Ninety-two percent of the specimens demonstrated at least one of the lytic EBV proteins. BZLF1 protein was seen in 81% of the specimens, whereas 54% and 52% of specimens expressed diffuse and restricted early antigens, respectively. Twenty-nine percent of specimens contained all three of the lytic proteins. The NotI tandem DNA probe produced staining in 88% of the specimens, whereas immediate early and late lytic transcripts were seen in 90% of the specimens. The amount of lytic activity did not significantly vary with PTLD histopathologic findings, but monomorphous proliferations localized to a single anatomic site showed a slightly lower reactivity with the antibodies and nucleic acid probes. Lytic activity was highest in patients with multisite disease, although this difference was not statistically significant. In conclusion, a significant proportion of patients with PTLD expressed lytic nucleic acids and proteins. Further work is necessary to analyze the role of lytic EBV infection in the initiation and maintenance of lymphoproliferative disorders in allograft recipients.