Tissue sections containing protozoa with birefringent spores indicate an infection by microsporidia. Hematoxylin and eosin (HE) does not affect spore birefringence, but some special stains (Goodpasture, Brown and Brenn, or Gram) obscure it. Encephalitozoon cuniculi from an infected puppy, Glugea stephani from the winter flounder Pseudopleuronectes americanus, and Plistophora sp. from the Japanese eel Anguilla japonica all have birefringent spores. Encephalitozoon was studied first and then the two genera from fishes were included for comparison. Small masses of newly formed spores (pseudocysts) line Glugea cysts and then merge into the contents of the cyst as it enlarges and bulges through the intestinal musculature to become subserosal. The birefringence of Plistophora is present in fully mature spores contained in pseudocysts, but may disappear when the spores are released and become involved in granulomas. Coccidians from various hosts were always nonbirefringent. Whenever a protozoan organism in a tissue could be either microsporidian or coccidian, a test for birefringence, if positive, resolves the question. There may be no need to use a special stain.