EBNA 1 was expressed as a nonfusion protein in Escherichia coli under control of the lac promoter. The major immunoreactive EBNA 1 proteins migrated as two doublets with molecular masses of about 39/41 and 49/51 kDa. Gel mobility shift experiments showed that these products exhibit the sequence-specific DNA binding on ori P previously characterized for a 28-kDa lambda N-fusion protein encompassing the carboxy third of the EBNA 1 protein. Three monoclonal antibodies previously found to react with EBNA 1 were shown to block binding of DNA by the EBNA 1 products expressed in bacteria. The same monoclonal antibodies also blocked specific DNA binding by EBNA 1 produced in Burkitt lymphoma cells infected by EB virus. Fab fragments of two monoclonal antibodies inhibited DNA binding by EBNA 1, indicating that the antibodies recognize an epitope of the protein that is involved in the recognition of DNA, or another domain crucial for DNA binding such as a dimerization site. Some but not all human antisera with antibody to EBNA 1 neutralized specific binding of EBNA 1 to DNA. These findings will help to map the residues of the EBNA 1 protein which are essential for specific binding of DNA.