A naturally occurring case of chronic mastitis caused by Prototheca zopfii was detected in a dairy cow. The cow was observed for 12 weeks, and milk samples were cultured each week. Milk production was decreased, and high numbers (greater than 10(3)/ml) of the organism were detected in the milk. Six glands of 2 cows were inoculated with small numbers of P zopfii. All 3 cows were euthanatized, and at postmortem, a progressive pyogranulomatous inflammation was observed in all infected mammary glands. The organism was recovered from tissue and secretions from all inoculated glands and from lymph nodes draining these glands but not from other tissues.