OBJECTIVE-To evaluate the usefulness of an antibody detection ELISA and protein electrophoresis (PE) for diagnosing Encephalitozoon cuniculi (ECUN) infection in pet rabbits. ANIMALS-203 pet rabbits. PROCEDURES-Serum and plasma samples from pet rabbits were submitted from veterinary clinics within the United States. Participating veterinarians completed a questionnaire that was used to classify rabbits as clinically normal (n=33), suspected of having an ECUN infection (103), or clinically abnormal but not suspected of having an ECUN infection (67). An ELISA for detection of serum or plasma IgG against ECUN was developed by use of commercially available reagents. Results of the ELISA and PE were used to detect ECUN infection. RESULTS-A high seroprevalence of antibody against ECUN was detected in all 3 groups of rabbits. In rabbits suspected of having an ECUN infection, the mean IgG titer was 1.7 times as high as the values in the other rabbit groups. Rabbits suspected of having an ECUN infection and those that were simply clinically abnormal had a higher concentration of gamma-globulins than clinically normal rabbits. This increase in globulins concentration was accompanied by a decrease in the albumin-to-globulin ratio. Results of the ELISA and PE were significantly different between clinically normal rabbits and those suspected of having an ECUN infection. CONCLUSIONS AND CLINICAL RELEVANCE-The combination of an ELISA and PE may aid in the diagnosis of ECUN infection in pet rabbits. IMPACT FOR HUMAN MEDICINE-Because ECUN is a potential zoonotic agent, diagnostic methods for pet rabbits need to be improved to protect human health.