Cytoplasmic RNA prepared from five lymphoid cell lines and a Burkitt lymphoma biopsy was radioactively labeled in vitro and hybridized to cloned EcoRI restriction endonuclease fragments of B95-8 Epstein-Barr virus DNA. The results confirmed that the most abundant cytoplasmic RNA species in such cells is specified by a small region of the genome defined by the EcoRI J fragment. Detailed mapping experiments precisely localized these transcripts within the sequence of the rightmost one-third of the EcoRI J fragment. DNA sequencing suggested that this region of the Epstein-Barr virus genome is unable to code for protein. The major early transcripts consisted of two non-polyadenylated RNA species, each about 170 nucleotides in length. They were both transcribed off the same strand of the DNA and showed significant sequence homology with each other. The coding sequences of the two small RNAs contained potential intragenic control regions for RNA polymerase III.