To develop and evaluate a polyvalent ELISA incorporating a highly specific recombinant antigen (p44) for diagnosis of granulocytic ehrlichiosis in dogs and horses. 32 dogs and 43 horses. Results of the ELISA were compared with results of indirect fluorescent antibody (IFA) staining and western immunoblotting incorporating whole-cell antigen. For the canine and equine samples, percentages of samples with positive IFA staining, western immunoblotting, and ELISA results were similar. For 29 (91 %) canine samples and 30 (70%) equine samples, results of IFA staining, western immunoblotting, and the ELISA were in complete agreement. Results of the ELISA for 3 canine serum samples known to contain antibodies to Ehrlichia canis and 12 equine serum samples known to contain antibodies to E risticii were negative. Results of the present study suggest that a polyvalent ELISA incorporating a recombinant p44 antigen is suitable for detecting antibodies to E equi in dogs and horses.