Summary Diffuse large B‐cell lymphoma (DLBCL), the most common type of malignant lymphoma, accounts for 30% of adult non‐Hodgkin lymphomas. Epstein–Barr virus (EBV) ‐positive DLBCL of the elderly is a newly recognized subtype that accounts for 8–10% of DLBCLs in Asian countries, but is less common in Western populations. Five DLBCL‐derived cell lines were employed to characterize patterns of EBV latent gene expression, as well as response to cytokines and chemotaxis. Interleukin‐4 and interleukin‐21 modified LMP1, EBNA1 and EBNA2 expression depending on cell phenotype and type of EBV latent programme (type I, II or III). These cytokines also affected CXCR4‐ or CCR7‐mediated chemotaxis in two of the cell lines, Farage (type III) and Val (type II). Further, we investigated the effect of EBV by using dominant‐negative EBV nuclear antigen 1(dnEBNA1) to eliminate EBV genomes. This resulted in decreased chemotaxis. By employing an alternative way to eliminate EBV genomes, Roscovitine, we show an increase of apoptosis in the EBV‐positive lines. These results show that EBV plays an important role in EBV‐positive DLBCL lines with regard to survival and chemotactic response. Our findings provide evidence for the impact of microenvironment on EBV‐carrying DLBCL cells and might have therapeutic implications. Epstein–Barr virus (EBV) plays an important role in EBV‐positive diffuse large B‐cell lymphoma lines with regard to survival and chemotactic response.