ABSTRACTSalmonellosis represents a worldwide health problem because it is one of the major causes of food-borne disease. Although motility is postulated as an importantSalmonellavirulence attribute, there is little information about variation in motility in natural isolates. Here we report the identification of a point mutation (T551 → G) inmotA, a gene essential for flagellar rotation, in severalSalmonella entericaserovar Enteritidis field isolates. This mutation results in bacteria that can biosynthesize structurally normal but paralyzed flagella and are impaired in their capacity to invade human intestinal epithelial cells. Introduction of a wild-type copy ofmotAinto one of these isolates restored both motility and cell invasiveness. ThemotAmutant triggered higher proinflammatory transcriptional responses than an aflagellate isolate in differentiated Caco-2 cells, suggesting that the paralyzed flagella are able to signal through pattern recognition receptors. A specific PCR was designed to screen for the T551 → G mutation in a collection of 266S. Enteritidis field isolates from a nationwide epidemic, comprising 194 from humans and 72 from other sources. We found that 72 of the 266 (27%) isolates were nonmotile, including 24.7% (48/194) of human and 33.3% (24/72) of food isolates. Among nonmotile isolates, 15 carried the T551 → G mutation and, significantly, 13 were recovered from food, including 7 from eggs, but only 2 were from human sources. These results suggest that the presence of paralyzed flagella may impair the ability ofS. Enteritidis to cause disease in the human host but does not prevent its ability to colonize chickens and infect eggs.