A 4.5 kb M. agalactiae DNA fragment present in strains from different areas of Sardinia was cloned and used to detect M. agalactiae DNA in sheep milk samples by dot blot hybridization. The probe recognized only M. agalactiae strains and did not cross-hybridize with other mycoplasmas (M. capricolum, M. mycoides subsp. capri, M. mycoides subsp. mycoides, M. putrefaciens and M. arginini) or bacteria (E. coli, P. hemolytica, S. aureus, S. epidermis, L. casei, S. durans, S. lactis and S. thermophilus) which may also be found in sheep milk.