SUMMARYThe prevalence ofSalmonellain four layer farms in eastern Japan was investigated between 2004 and 2006 to determine the role of roof rats (Rattus rattus) in the epizootology ofSalmonella entericasubsp.entericaserovar Enteritidis (S. Enteritidis). PersistentS. Enteritidis andS. Infantis contamination of the environment and pooled egg samples were detected in three out of four layer farms. A total of 113 (13·3%) and 158 (18·6%) out of 851 rats examined were positive forS. Enteritidis andS. Infantis, respectively. By pulsed-field gel electrophoresis, only one indistinguishable pulsed-field pattern was yielded byS. Enteritidis strains from rats, eggs and environmental samples from each of the two contaminated layer farms. Although, a variety of pulsed-field patterns were generated byS. Enteritidis isolates from rats, eggs, and the environment of the other contaminated farms, there are, however, someS. Enteritidis strains that are closely related clones. These results suggest that roof rats are carriers ofS. Enteritidis andS. Infantis and that persistentS. Enteritidis andS. Infantis infections in a rat population may play an important role in the spread and maintenance of these pathogens inside the layer premises.