A substantial increase in the prevalence of S. enterica serovar Tennessee was observed in broiler flocks in Denmark at the turn of the year 1994 and in the following months. Epidemiological data indicated that a single hatchery was involved in spreading of the infection. Molecular characterization of S. enterica serovar Tennessee isolates from Danish broilers (1992 to 1995), the suspected hatchery and strains from various other sources included for comparison was initiated in order to trace the source of infection of the broilers. In general, strains of S. enterica ser. Tennessee showed only minor genotypic variation. Three different ribotypes were demonstrated when EcoRI was used for digestion of DNA. Two types were obtained by the use of HindIII. Nine different plasmids and seven different plasmid profiles were demonstrated. A 180 kb plasmid was, however, only demonstrated in isolates from broilers and the hatchery. Sixty-nine per cent of the broiler isolates obtained during the period 1992 to 1995 harboured this plasmid and 88% of the hatchery isolates contained a plasmid of the same size. An increased number of the broiler isolates (79%) contained this plasmid at the turn of 1994. Restriction enzyme analysis of the plasmid ensured that the plasmids from broilers and the hatchery were identical. By analysis of cleaning and disinfection procedures and by sampling of different control points in the hatchery it was shown that S. enterica ser. Tennessee had colonized areas of the hatchers which were protected from routine cleaning and disinfection. Subsequent inclusion of these areas into the sanitation programme resulted in the elimination of S. enterica ser. Tennessee from the hatchers, and a decreasing prevalence of S. enterica ser. Tennessee was observed in broiler flocks during the following months.