To assess the importance of TonB-dependent iron transport systems to growth of Shigella in vivo, a tonB mutant of Shigella dysenteriae was isolated and tested in cultured cells. The tonB mutant invaded epithelial cells, but did not form plaques in confluent monolayers of Henle cells, indicating an inability of this mutant to spread from cell to cell. The rate of intracellular multiplication of the tonB mutant was reduced significantly compared to that of the wild type. The loss of virulence in the tonB mutant was not due to loss of either Shu or Ent, the TonB-dependent systems which allow for transport of heme and ferrienterobactin, respectively. A shuA mutant lacking the outer membrane receptor for heme, an entB mutant defective in enterobactin synthesis, and a shuA entB double mutant each were able to invade cultured cells, multiply intracellularly, and form wild-type plaques. The ability of S. dysenteriae to access iron during intracellular growth was assessed by flow cytometric analysis of an iron- and Fur-regulated shuA-gfp reporter construct. Low levels of green fluorescent protein expression in the intracellular environment were observed in all strains, indicating that iron is available to intracellular bacteria, even in the absence of TonB-dependent iron transport. The failure of the tonB mutant to grow well in an iron-replete intracellular environment suggests that TonB plays a role in addition to heme- and siderophore-mediated iron acquisition in vivo, and this function is required for the intracellular growth and intercellular spread of S. dysenteriae.