The extrachromosomal Epstein-Barr virus (EBV) plasmid in the Burkitt lymphoma cell line, Raji, is stably associated with the nuclear matrix. This association is effected by a nuclear matrix attachment region (MAR) located in the BamHI C fragment of the viral genome; no other region of EBV DNA was found to be attached to the nuclear matrix with high affinity. The MAR was mapped to 5.2 kbp of DNA, greater than 80% of which is found on the nuclear matrix in unsynchronized cells expressing only viral latent cycle products. Thus the majority of viral plasmids in Raji cells use the same MAR. The MAR of EBV DNA contains the origin of latent viral DNA replication (oriP), the genes for the small viral RNAs (EBERs) and a 500 bp region immediately upstream of the EBER-1 gene. The clustering of the latent viral replication origin and the nearby enhancer and promoters for latent viral transcription on the nuclear matrix is likely to be crucial for regulation of the latent viral genome.