Canine factor VIII (FVIII) preparations isolated from cryoprecipitates by gel chromatography were pooled to provide 1 batch of antigen for simultaneous immunization of 2 rabbits and a goat. The goat and rabbit antisera had similar FVIII-neutralizing titers, but the latter had 7-10 times more precipitating titer for FVIII-related antigen (FVIII-RA). Absorption with material low in FVIII had little effect on the precipitating titer of the rabbit antibody, but it abolished the precipitating capacity of the goat antibody and caused a 20% reduction in the neutralizing titer of both antisera. Results obtained in the Laurell assay with the 2 different antisera were similar. This finding was true whether the FVIII-RA levels were reduced, normal, or elevated, as well as for heat-treated and frozen-thawed plasmas. Both antisera were neutralized by the same canine plasma to a similar extent. Analysis of FVIII concentrates by crossed immunoelectrophoresis suggested that canine FVIII-RA was heterogeneous, with slow- and fast-migrating components. The presence of more than 1 antigenic site on the FVIII complex was also supported by the disparity between the FVIII-neutralizing and -precipitating titers of goat antiserum and by the demonstration that FVIII-RA, FVIII-neutralizing antigen and procoagulant activity varied independently.