Using RT-PCR analysis of Epstein-Barr virus (EBV) latent gene transcription in EBV-harboring cell lines (JY and RAJI) and in post-transplantation lymphoproliferative disorders (PT-LPDs), we detected transcription of all tested latent genes (EBNA1, EBNA2, LMP1, LMP2A, and BARF0) in all cases, suggesting the presence of similar EBV expression patterns in both PT-LPDs and cell lines. In addition, the detection of immediate early (ZEBRA) and early gene (BHRF1) transcripts in cell lines and PT-LPDs indicates that activation of the virus lytic cycle occurs. To investigate EBV expression patterns at the single-cell level, a combination of immunohistochemistry and RNA in situ hybridization (including double-staining procedures) was used. In the JY and RAJI cell lines, the latency type 3 expression pattern was detected in 80 to 90% of the cells as shown by the co-expression of EBNA2 and LMP1. In contrast, in the three PT-LPDs that could be analyzed by double staining, cells expressing both EBNA2 and LMP1 were rarely detected. A mixture of at least three different cell populations were identified: (1) cells exclusively expressing EBER1/2 and EBNA1 (latency type 1); (2) cells expressing EBER1/2, EBNA1, and LMP1 (latency type 2); and (3) cells expressing EBER1/2, EBNA1, and EBNA2 in the absence of LMP1. Activation of the lytic cycle was observed in a small minority of cells, as demonstrated by detection of ZEBRA and EA-D in all cases and GP350/220 in two cases. Thus, in contrast to EBV-transformed cell lines, the observed EBV gene expression pattern in PT-LPDs reflects a mixture of multiple EBV-harboring subpopulations expressing different subsets of EBV-encoded proteins. These data indicate that the operational definitions of EBV latencies in vitro cannot easily be applied to PT-LPDs but that a continuum of different latency expression patterns can be detected at the single cell level in these lymphomas with, in a small minority of cells, progression to the virus lytic cycle.