We recently generated a monoclonal antibody immunoglobulin G1 (MAb 10G5), which can recognize 15- to 17-kDa antigens, virulence-associated antigens of Rhodococcus equi, and developed a colony blot enzyme-linked immunosorbent assay with MAb 10G5 for the rapid identification of virulent R. equi. In this epidemiologic study, we evaluated the results of the colony blot test in the identification of virulent isolates of R. equi from feces of horses and soil and compared them with those from a conventional procedure (plasmid profiles of isolates by agarose gel electrophoresis). Environmental isolates (778 isolates from feces of foals, 170 isolates from feces of dams, and 1, 267 isolates from soil on horse-breeding farms in Hokkaido) were tested by the colony immunoblot test, and 238 of the 778 isolates, 6 of the 170 isolates, and 85 of the 1, 267 isolates showed positive signals. Positive isolates were then analyzed for the presence of virulence plasmid DNA, and 235 (98.7%) of the 238 isolates from foals, 6 (100%) of the 6 isolates from dams, and 75 (88.2%) of the 85 isolates from soil showed the presence of virulence plasmids. On the other hand, 50 isolates from each source, which were randomly selected from the isolates that showed negative signals by colony immunoblot, did not contain virulence plasmids. These results demonstrated that the colony blot test that uses a monoclonal antibody specific for virulence-associated antigens is a rapid and reliable test for the identification of virulent R. equi.