Five types of anti-VP35 monoclonal antibodies (MAbs), four immune sera against Marburg virus (MBGV), and 11 overlapping recombinant VP35 fragments were used to map the epitopes for VP35 of MBGV. The purified full-size recombinant VP35 was highly immunogenic and retained the B-cell epitopes that were identical to those of the viral VP35. Two major sites on VP35 and a set of truncated VP35 fragments were found by use of an enzyme immunoassay and immunoblot. Site I was located in a region between amino acids 1 and 174 of the VP35 sequence, and only polyclonal antibodies (PAbs) against MBGV recognized epitopes at this site. Site II was mapped by use of anti-VP35 MAbs to the region between amino acid residues 167 and 278 of VP35. Amino acids 252-278 of VP35 might be involved in the formation of the epitopes for MAbs. B-cell epitopes were not found on the C-terminus of VP35 by use of PAbs or MAbs.