Specific binding sites for Epstein-Barr virus (EBV) nuclear antigen (EBNA-1), isolated and semipurified from EBV-transformed nonproductive Raji cells, were visualized on the molecule of EBV DNA by electron microscopy and mapped. Two measures had to be applied to counteract the limited purity of the EBNA-1 preparation: (i) EBV DNA/EBNA-1 complexes were specifically enlarged by binding with anti-EBNA-1 (IR-3) IgG antibody. (ii) DNA-binding proteins that did not react with the anti-EBNA-1 antibody were eluted from EBV DNA with 1.5 M NaCl, taking advantage of the resistance of DNA/EBNA-1/anti-EBNA-1 antibody complexes to the high-salt treatment. EBNA-1 bound at the highest relative frequency (greater than 30%) to the EBV DNA map positions of 8.8 +/- 0.3, 10.3 +/- 0.5, and 46.6 +/- 1.2 kb. It bound with a lower but still statistically significant frequency (16%) to the map positions of 64.5 +/- 1.0, 89.7 +/- 1.6, 129.6 +/- 1.1 kb.