The clinical, epidemiological and pathological aspects of protothecal mastitis in a Brazilian dairy herd are described. Prototheca zopfii infection was diagnosed in 11 of 121 milking cows. Clinical mastitis refractory to usual therapy was observed in 7 cows. Several environmental conditions conducive to the growth of Prototheca spp., such as wetness, muddiness and the presence of organic material, were present in the dairy. Improper milking practices and insanitary infusion of the intramammary antibiotics were also observed. Six cows with protothecal mastitis were slaughtered and the affected quarters of each cow were examined by histology and immunohistochemical staining for bovine keratin and P. zopfii. The histological lesions were characterized by interstitial infiltrates of macrophages, plasma cells and lymphocytes; algae were seen in the alveolar lumen and interstitium. The lack of a positive reaction with an antiserum against bovine keratin in the mammary alveolar epithelial layer in some affected areas suggests destruction of milk-producing tissues, which may be related to the low milk production observed. The algal organisms stained positively with a polyclonal antibody against P. zopfii.