Thirty human and nonhuman primate sera tested at the Centers for Disease Control by enzyme-linked immunosorbent assay (ELISA), immunofluorescent antibody assay (IFA), and Western blotting were retested at the Virus Reference Laboratory, Inc. by the dot-immunobinding assay (DIA). The Ebola-Reston strain of virus received from the Centers for Disease Control was prepared into a suitable DIA antigen as described for other antigens. All six Western blotting-positive sera were also positive by DIA, as were the five ELISA-positive sera. Testing by IFA, the original test of choice, indicated an additional four seropositives, all negative by the other test systems. Of 288 randomly selected macaque sera, 19 were also found to be Ebola-Reston virus-positive by DIA.