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Rabbit antibodies against Encephalitozoon cuniculi were detected in an enzyme immunoassay procedure in which antigen was grown and used in situ. The test appeared to be more sensitive than the indirect immunofluorescence test with which it was compared, but gave essentially the same results for the 64 sera evaluated. This procedure will allow any laboratory with cell-culture facilities to produce a diagnostic antigen without the need for antigen processing. It is simple and reliable, and does not require specialized equipment or microscopic assessment.