Salmonella Enteritidis is a major food-borne pathogen that causes nontyphoidal diarrhoea in humans. Infection of adult egg-laying hens usually results in symptomless carriage but in young chicks it may cause paratyphoid disease. It is not known whether S. Enteritidis requires genes additional to known virulence genes for systemic infection of young chickens. A transposon insertion library was created using S. Enteritidis 10/02, which yielded 1246 mutants. Of 384 mutants screened in chickens for attenuation (30.8% of insertion library), 12 (3.1%) had a 50% lethal dose at least 100 times that of the parental strain. Sequencing revealed insertions in genes involved in the biosynthesis of lipopolysaccharide, cell membrane, ATP biosynthesis, transcriptional regulation of virulence and the yhbC gene, which has an unknown function. Evaluation of in vitro virulence characteristics of a Delta yhbC mutant revealed that its ability to invade HeLa cells and survive within a chicken macrophage cell line (HD11) was significantly reduced. It was also less resistant to reactive oxygen and nitrogen intermediates and had a retarded growth rate. Chickens challenged with the Delta yhbC mutant cleared the organism from the liver and spleen 1 week faster than the parental strain and were able to develop specific serum IgG antibodies against the Delta yhbC mutant.