Paratuberculosis, a naturally occurring disease of domestic and wild ruminants, has been reported in cattle, sheep and goats in Italy. Water buffalo (Bubalus bubalis) is another economically important species in our Country, especially in Latium (Central Italy ) and Campania (Southern Italy). Following obtainment of a financial aid on a research project aimed to animal productions' improvement, which has been issued by the European Union (5b Project), 15 water- buffalo herds from three different provinces of Latium region (Frosinone, Latina, Rome) were investigated between 1997 and 1998. More in detail, a total number of 1,321 animals were submitted to serological tests for M.paratuberculosis antibody detection (AGID and ELISA). Three adult subjects, all belonging to an identical farm, turned out to be positive. Only one of these, a 5- years-old female, showed clinical signs related to the disease, namely progressive emaciation, weakness, chronic intermittent diarrhoea, reduced milk production, and infertility. The disease started in November 1997 and a second blood sampling was carried out 7 months later on all three concerned subjects, the remaining two of which showed, however, no evidence of clinical disease. At the same time, faecal samples were collected from the three animals. Detection of several aggregates of acid-fast bacilli with mycobacterial morphology was achieved only in the clinically affected subject, which is still alive. On the other hand, all three investigated buffaloes remained seropositive. Microbiological investigations for M. paratuberculosis on faecal samples have so far yielded negative results. To our knowledge, this is the first case of M. paratuberculosis infection in water buffaloes from Italy.