Convalescent swine serum to African swine fever virus (ASFV) isolate E75 neutralized the infectivity of virulent ASFV isolates E75, E70, Lisbon 60, Malawi Lil 20/1 and a low passage tissue culture adapted variant of E75, E75CV/V3, by 86-97% in Vero and macrophage cell cultures. A monoclonal antibody, mAb-135D4, recognizing an ASFV protein of 72 kDa also exhibited strong neutralizing activity with these viruses. Unexpectedly, both E75 immune sera and mAb-135D4 failed to neutralize high passage tissue culture adapted ASFV variants including Lisbon 60, Haiti, Dominican Republic I, Dominican Republic II, and Brazil II, even though mAb-135D4 reacted with all viruses. These results suggest that tissue culture adaptation of ASFV isolates may be associated with loss of specific determinants associated with virus neutralization. To our knowledge, this is the first report of an ASFV neutralizing epitope.