Hemadsorption (Had) of erythrocytes to the surface of African swine fever virus (ASFV)-infected cells is a well-known phenomenon but hemagglutination of pig erythrocytes in the supernatant of ASFV-infected cells has not been reported before. We report here the discovery of a pig erythrocyte-agglutinating activity released to the in vitro cell culture medium by cells infected with some isolates of ASFV. This finding allowed the identification and characterization of a soluble hemagglutinin (HA) molecule that could be separated from the ASFV particles either by ultracentrifugation or by gel-permeation chromatography. The HA was inactivated by agents known to affect protein conformation such as heat, beta-mercaptoethanol, urea, and guanidine isothiocyanate. Glycosylation seemed to be of importance since treatment of HA with glycosidase F inhibited the hemagglutinating activity and HA could be partially purified by affinity chromatography on immobilized concanavalin A. When native it had an estimated molecular weight of 300 kDa by gel-permeation chromatography yielding 51-kDa protein monomers under denaturing conditions as identified by immunoblotting. Preliminary attempts to correlate the induced anti-HA serum antibodies with viremia or infection-inhibition serum antibodies after infection of pigs with attenuated ASFV or immunization with purified HA are also reported.