EBNA-2 is the first protein to be detected after infection of primary B lymphocytes by Epstein-Barr virus (EBV) and plays an essential role as transcriptional activator in EBV-induced lymphocyte transformation. We analysed by PCR and sequencing regions of the EBNA-2 type 1 gene from isolates from 13 children with infectious mononucleosis (IM), 6 children with tonsillar hyperplasia (TH), and 9 patients with HIV infection followed longitudinally. We found in all three groups of patients frequent non-silent point mutations at positions 48990, 48991, 49021, 49057, 49083, 49089, 49091, 49113, 49119, 49140, 49156, and a triplet insertion at position 49136. While 4 out of 13 samples from patients with IM showed a mosaic pattern suggesting co-existence of more than 1 substrain of EBNA-2 type 1, none of the samples from TH showed this pattern consistent with substrain selection during clinical latency. No sequence changes were noted over time in samples derived from patients with HIV infection. We conclude that in analogy to the coexistence of several subtypes of EBNA-1 in healthy EBV carriers, samples from IM can harbor more than one subtype of the EBNA-2 type 1 gene.