Functions of the six Epstein-Barr virus latent infection nuclear proteins (EBNA-1, -2, -3A, -3B, -3C, or -LP) in maintaining latent infection or cell growth transformation are only partially understood. Using antibodies specific for each EBNA in immunofluorescence microscopy, EBNA-2, -3A, and -3C localized to subnuclear granules which fill much of the nucleus, excluding nucleoli. EBNA-LP localized to a small number of discrete subnuclear particles, also excluding nucleoli. Only EBNA-1 associated with metaphase chromosomes. Concordantly, in biochemical nuclear fractionation studies, EBNA-1 was the major chromatin-associated EBNA. EBNA-1 also differed from the other EBNAs in the extent of its association with the nucleoplasm and in its lack of nuclear matrix association. EBNA-LP, -2, -3A, and -3C were associated with the nuclear matrix, although they were also found in the nucleoplasm and to a lesser extent in the chromatin fractions. Metabolic 32Pi-labeling of cells followed by two-dimensional gel electrophoresis showed that EBNA-LP could be resolved into multiple phosphorylated isoforms. EBNA-2 was also phosphorylated and many isoforms were detected by isoelectric focusing.