The lytic cycle-associated lytic latent membrane protein-1 (lyLMP-1) of Epstein-Barr virus (EBV) is an amino-terminally truncated form of the oncogenic LMP-1. Although lyLMP-1 shares none of LMP-1's transforming and signal transducing activities, we recently reported that lyLMP-1 can negatively regulate LMP-1-stimulated NF-kappaB activation. The lyLMP-1 protein encoded by the B95-8 strain of EBV initiates from methionine 129 (Met129) of the LMP-1 open reading frame (ORF). The recent report that Met129 in the B95-8 LMP-1 ORF is not conserved in the Akata strain of EBV prompted us to screen a panel of EBV-positive cell lines for conservation of Met129 and lyLMP-1 expression. We found that 15 out of 16 tumor-associated virus isolates sequenced encoded an ATT or ACC codon in place of ATG in the LMP-1 ORF at position 129, and tumor cell lines harboring isolates lacking an ATG at codon 129 did not express the lyLMP-1 protein. In contrast, we found that EBV DNA from 22 out of 37 healthy seropositive donors retained the Met129 codon. Finally, the lyLMP-1 initiator occurs variably within distinct EBV strains and its presence cannot be predicted by EBV strain identity. Thus, Met129 is not peculiar to the B95-8 strain of EBV, but rather can be found in the background of several evolutionarily distinct EBV strains. Its absence from EBV isolates from tumors raises the possibility of selective pressure on Met129 in EBV-dependent tumors.