Precipitin lines not associated with equine infectious anemia (EIA) were observed in routine agar gel immunodiffusion (AGID) testing for the infection. The serums which produced these lines were obtained from horses which had been given multiple vaccinations with commercially available cell culture-origin equine virus vaccines as part of a comprehensive herd health program. The lines formed against cell culture-derived, but not spleen-derived EIA viral antigens. Investigation revealed that bovine serum proteins in the vaccines induced precipitating antibodies which reacted with bovine serum proteins in cell culture-derived antigens. A vaccination trial, utilizing 4 commercially available vaccines in various combinations, indicated that as few as 2 vaccinations could induce AGID-detectable antibodies to bovine serum proteins in individual ponies. These antibodies were very transitory, usually lasting no longer than a week. Some horses, however, which had been given 4 vaccinations developed similar antibodies which persisted 3 months beyond the last vaccination. The extraneous precipitin lines produced by these antibodies in the AGID test for EIA were readily distinguished from true EIA-associated reactions and did not result in false-positive interpretations of the test. However, heavy percipitin lines due to strong antibovine serum activity did mask weakly positive EIA reactions.