Methods available for the diagnosis of Ehrlichia phagocytophila infection are limited and of low sensitivity. Since polymerase chain reaction (PCR) is a highly sensitive and specific method for the detection of infectious agents in animals, it was considered useful to develop a PCR method specific for this agent. An experimental protocol in which blood from 10 different flocks was inoculated into 10 naive 1 month-old lambs was carried out. Progress of the infection was monitored during 3 weeks by daily rectal temperature and blood samples. Eight lambs showed hyperthermia, presence of E. phagocytophila in blood, and lower growth rate while the other two never showed the agent in their blood or any clinical signs. This experimental infection, essentially designed to provide infected samples, also showed that flocks from areas previously thought to be free of E. phagocytophila were actually infected. PCR on blood samples allowed detection of the infection earlier than in case of microscopic examination. This was related to an increased sensitivity (87-91%). PCR yielded positive results on samples with as few as 1.08 infected neutrophils/µl (about 0.1%).