A dairy farm that has been suffered continuously(more than 2 years) from brucellosis in Korea in spite of repeated legal test-and-slaughter was investigated the main source of infection in the farm. All cattle(22 milking cows, 44 heifers, 60 calves, 8 bull), dogs(3 mixed breed), feces from wild birds(3 samples), drinking water(3 sites), and soil in the paddocks(14 sites) inside the farm were examined with serological and/or bacteriological methods including specific DNA detection with PCR method. Brucella spp in the milk and blood were detected in 12/22 and 5/22 milking cows, respectively, although all of them were negative with conventional tube agglutination test. The number of serologically positive heifer was 15(15/44), but the isolation of Brucella spp was succeeded in the only 11(11/15) of them. Brucella were detected in vagina 1(1/11) and nasal(3/12) excretion in serologically positive heifers. All the three dogs were serologically positive, and Brucella spp were isolated from their blood. However, Brucella spp were not detected in the drinking water, soil in the paddocks, nor the feces of wild birds. The results suggest that milking cow secrete Brucella spp through milk, genital tract and nasal cavity, which are the major source of infection in this farm, The main infection route of Brucella spp is contact to contact with Brucella spp excreting animals rather than environmental contamination. The animals, living together with infected cow such as dogs, are the readily susceptible and are required to be examined for Brucella spp.